SOLUBLE FMS-LIKE TYROSINE KINASE-1 (sFLT-1) ANTIBODY AND RELATED COMPOSITION, KIT, METHODS OF USING, AND MATERIALS AND METHOD FOR MAKING

ABSTRACT

An isolated antibody that specifically binds to sFlt-1 or a fragment thereof having (i) a variable heavy domain region comprising the amino acid sequence of SEQ ID NO: 2, (ii) a variable light domain region comprising the amino acid sequence of SEQ ID NO: 4, or (iii) both (i) and (ii), a pharmaceutical composition and a kit comprising such an antibody, a method of making such an antibody, a method of determining the presence, amount or concentration of sFlt-1 or a fragment thereof in a test sample, a method of treating a patient in therapeutic or prophylactic need of an antagonist of sFlt-1, an isolated nucleic acid comprising a nucleotide sequence encoding the amino acid sequence of (i) SEQ ID NO: 2, (ii) SEQ ID NO: 4, or (iii) both (i) and (ii), optionally as part of a vector, and a host cell comprising and expressing such a nucleic acid.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of copending U.S. application Ser. No.12/643,847, filed Dec. 21, 2009, which claims the priority of U.S.Provisional Patent Application 61/140,561, filed on Dec. 23, 2008, thedisclosures of which applications are incorporated herein by referencein their entireties.

TECHNICAL FIELD

This disclosure relates to an antibody, an isolated nucleic acid,optionally as part of a vector, a host cell comprising same, a method ofmaking an antibody, a method and a kit for determining the presence,amount or concentration of an analyte in a sample, a pharmaceuticalcomposition comprising an antibody, and a method of using thecomposition to treat a patient.

BACKGROUND

Preeclampsia is a syndrome of hypertension, edema, and proteinuria. Itaffects about 5-10% of pregnant women, and results in substantialmaternal and fetal morbidity and mortality. Symptoms of preeclampsiatypically appear after the 20^(th) week of pregnancy and are usuallydetected by routine screening of the pregnant woman's blood pressure andurine. Such routine screening methods, however, are ineffective forearly diagnosis.

FMS-like tyrosine kinase-1 (Flt-1) is a membrane-spanning tyrosinekinase receptor that is differentially expressed in endothelial cells.It is highly expressed by trophoblast cells, which contribute to theformation of the placenta during pregnancy. Vascular endothelial growthfactor (VEGF), which is an endothelial cell-specific mitogen, anangiogenic inducer, and a mediator of vascular permeability, binds as ahomodimer to Flt-1. Placental growth factor (P/GF) is a member of theVEGF family that also is involved in development of the placenta. P/GFis expressed by cytotrophoblasts and syncytiotrophoblasts, can induceproliferation, migration, and activation of endothelial cells, and bindsas a homodimer to Flt-1. Thus, VEGF and P1GF both contribute tomitogenic activity and angiogenesis during development of the placenta.

sFlt-1 is a soluble form of the Flt-1 receptor, and, thus, is alsoreferred to as soluble VEGF receptor-1. It is a splice variant of theFlt-1 receptor that lacks the transmembrane and cytoplasmic domains ofthe Flt-1 receptor but contains seven IgG-like domains of the externalportion of the receptor Like the Flt-1 receptor, sFlt-1 binds to VEGFand P1GF; however, it does not stimulate mitogenesis of endothelialcells. Rather, sFlt-1 prevents proteins from initiating blood vesselgrowth. It has been identified as a biomarker for the diagnosis ofpreeclampsia (see, e.g., McKeeman et al., Amer. J. of Obstetrics andGynecology 191: 1240-1246 (2004)).

Immunoassays, such as enzyme-linked immunosorbent assays (ELISAs), havebeen developed to measure free and sFlt-1 complexed with VEGF in fluidsamples (see, e.g., Karumanchi et al., U.S. Pat. No. 7,335,362, whichissued Feb. 26, 2008, and Belgore et al., Clin. Sci. 100: 567-575(2001)). There remains a need, however, for new materials, methods, andkits for determining the concentration of sFlt-1 in a test sample, suchas in the diagnosis of preeclampsia and cardiovascular disease. Therealso is a need for new compositions and methods for treatment of apatient in therapeutic or prophylactic need of an antagonist of sFlt-1,such as a patient at risk for or having preeclampsia or a cardiovasculardisease. The present disclosure seeks to address such needs. These andother objects and advantages of the present disclosure will becomeapparent from the detailed description provided herein.

SUMMARY

An isolated antibody that specifically binds to soluble FMS-liketyrosine kinase-1 (sFlt-1) or a fragment thereof is provided. Theantibody has (i) a variable heavy domain region comprising the aminoacid sequence of SEQ ID NO: 2, (ii) a variable light domain regioncomprising the amino acid sequence of SEQ ID NO: 4, or (iii) a variableheavy domain region comprising the amino acid sequence of SEQ ID NO: 2and a variable light domain region comprising the amino acid sequence ofSEQ ID NO: 4.

An isolated antibody in one aspect specifically binds to solubleFMS-like tyrosine kinase-1 (sFlt-1) or a fragment thereof and has atlast one binding constant selected from the group consisting of anassociation rate constant (k_(a)) between about 9.0×10⁵ M⁻¹ s⁻¹ to about4.0×10⁶ M⁻¹ s⁻¹, a dissociation rate constant (k_(d)) between about1.0×10⁻⁴ s⁻¹ to about 6.0×10⁻⁴ s⁻¹ and an equilibrium dissociationconstant (K_(D)) between about 0.5×10⁻¹⁰ M to about 4.0×10⁻¹⁰ M.

A method of determining the presence, amount or concentration of sFlt-1or a fragment thereof in a test sample is also provided. The methodcomprises assaying the test sample for sFlt-1 (or a fragment thereof) byan immunoassay employing at least one antibody and at least onedetectable label. The method further comprises comparing a signalgenerated by the detectable label as a direct or indirect indication ofthe presence, amount or concentration of sFlt-1 (or a fragment thereof)in the test sample to a signal generated as a direct or indirectindication of the presence, amount or concentration of sFlt-1 (or afragment thereof) in a control or calibrator. The calibrator isoptionally part of a series of calibrators in which each of thecalibrators differs from the other calibrators in the series by theconcentration of sFlt-1 (or a fragment thereof). At least one antibodyis an isolated antibody, which specifically binds to sFlt-1 (or afragment thereof) and which has (i) a variable heavy domain regioncomprising the amino acid sequence of SEQ ID NO: 2, (ii) a variablelight domain region comprising the amino acid sequence of SEQ ID NO: 4,or (iii) a variable heavy domain region comprising the amino acidsequence of SEQ ID NO: 2 and a variable light domain region comprisingthe amino acid sequence of SEQ ID NO: 4. The method can be adapted foruse in an automated system or a semi-automated system.

The method can comprise the following steps: (i) contacting the testsample with at least one capture antibody, which binds to an epitope onsFlt-1 (or a fragment thereof), so as to form a capture antibody/sFlt-1(or a fragment thereof) complex, (ii) contacting the captureantibody/sFlt-1 (or a fragment thereof) complex with at least onedetection antibody, which comprises a detectable label and binds to anepitope on sFlt-1 (or a fragment thereof) that is not bound by thecapture antibody, to form a capture antibody/sFlt-1 (or a fragmentthereof)/detection antibody complex, and (iii) determining the presence,amount or concentration of sFlt-1 (or a fragment thereof) in the testsample based on the signal generated by the detectable label in thecapture antibody/sFlt-1 (or a fragment thereof)/detection antibodycomplex formed in (ii).

Alternatively, the method can comprise the following steps: (i)contacting the test sample with at least one capture antibody, whichbinds to an epitope on sFlt-1 (or a fragment thereof) so as to form acapture antibody/sFlt-1 (or a fragment thereof) complex, andsimultaneously or sequentially, in either order, contacting the testsample with detectably labeled sFlt-1 (or a fragment thereof), which cancompete with any sFlt-1 (or a fragment thereof) in the test sample forbinding to the at least one capture antibody, wherein any sFlt-1 (or afragment thereof) present in the test sample and the detectably labeledsFlt-1 compete with each other to form a capture antibody/sFlt-1 (or afragment thereof) complex and a capture antibody/detectably labeledsFlt-1 (or a fragment thereof) complex, respectively, and (ii)determining the presence, amount or concentration of sFlt-1 in the testsample based on the signal generated by the detectable label in thecapture antibody/detectably labeled sFlt-1 (or a fragment thereof)complex formed in (ii).

The method can further comprise simultaneously or sequentially, ineither order, determining the amount or concentration of vascularendothelial growth factor (VEGF) (or a fragment thereof) and/orplacental growth factor (P1GF) (or a fragment thereof) in the testsample, which method comprises assaying the test sample for VEGF (or afragment thereof) and/or P1GF (or a fragment thereof) by an assayemploying at least one specific binding partner for VEGF (or a fragmentthereof) and/or at least one specific binding partner for P1GF (or afragment thereof), respectively, and at least one detectable label andcomprising comparing a signal generated by the detectable label as adirect or indirect indication of the amount or concentration of VEGF (ora fragment thereof) and/or P1GF (or a fragment thereof) in the testsample to a signal generated as a direct or indirect indication of theconcentration of VEGF and/or P1GF, respectively, in a control orcalibrator, which is optionally part of a series of calibrators in whicheach of the calibrators differs from the other calibrators in the seriesby the amount or concentration of VEGF or P1GF, respectively.

The method can further comprise diagnosing, prognosticating, orassessing the efficacy of therapeutic/prophylactic treatment of apatient from whom the test sample was obtained. If the method furthercomprises assessing the therapeutic/prophylactic treatment of thepatient, the method optionally further comprises modifying thetherapeutic/prophylactic treatment of the patient as needed to improveefficacy.

Also provided is a kit for assaying a test sample for sFlt-1 (or afragment thereof). The kit comprises at least one component for assayingthe test sample for sFlt-1 (or a fragment thereof) and instructions forassaying the test sample for sFlt-1 (or a fragment thereof). At leastone component includes an isolated antibody that specifically binds tosFlt-1 (or a fragment thereof). The antibody has (i) a variable heavydomain region comprising the amino acid sequence of SEQ ID NO: 2, (ii) avariable light domain region comprising the amino acid sequence of SEQID NO: 4, or (iii) a variable heavy domain region comprising the aminoacid sequence of SEQ ID NO: 2 and a variable light domain regioncomprising the amino acid sequence of SEQ ID NO: 4. The antibody isoptionally detectably labeled.

A pharmaceutical composition is also provided. The composition comprisesa therapeutically or prophylactically effective amount of an isolatedantibody that specifically binds to sFlt-1 (or a fragment thereof). Theantibody has (i′) a variable heavy domain region comprising the aminoacid sequence of SEQ ID NO: 2, (ii′) a variable light domain regioncomprising the amino acid sequence of SEQ ID NO: 4, or (iii′) a variableheavy domain region comprising the amino acid sequence of SEQ ID NO: 2and a variable light domain region comprising the amino acid sequence ofSEQ ID NO: 4. The composition further comprises a pharmaceuticallyacceptable carrier, diluent, and/or excipient and, optionally, anotheractive agent and/or an adjuvant. The pharmaceutical composition isoptionally part of a kit comprising one or more containers in which theantibody, another active agent and/or the adjuvant can be present in thesame or different containers.

Also provided is a method of treating a patient in therapeutic orprophylactic need of an antagonist of sFlt-1. The method comprisesadministering to the patient a pharmaceutical composition comprising (i)a therapeutically or prophylactically effective amount of an isolatedantibody that specifically binds to sFlt-1 or a fragment thereof. Theantibody has (i′) a variable heavy domain region comprising the aminoacid sequence of SEQ ID NO: 2, (ii′) a variable light domain regioncomprising the amino acid sequence of SEQ ID NO: 4, or (iii′) a variableheavy domain region comprising the amino acid sequence of SEQ ID NO: 2and a variable light domain region comprising the amino acid sequence ofSEQ ID NO: 4. The composition further comprises a pharmaceuticallyacceptable carrier, diluent, and/or excipient and, optionally, anotheractive agent and/or an adjuvant.

An isolated nucleic acid is further provided. The nucleic acid comprisesa nucleotide sequence encoding the amino acid sequence of (i) SEQ ID NO:2, (ii) SEQ ID NO: 4, or (iii) SEQ ID NO: 2 and SEQ ID NO: 4, optionallyas part of a vector. The nucleic acid can comprise the nucleotidesequence of (i) SEQ ID NO: 1, (ii) SEQ ID NO: 3, or (iii) SEQ ID NO: 1and SEQ ID NO: 3, optionally as part of a vector.

Still further provided is a host cell. The host cell comprises andexpresses an isolated nucleic acid comprising a nucleotide sequenceencoding the amino acid sequence of (i) SEQ ID NO: 2, (ii) SEQ ID NO: 4,or (iii) SEQ ID NO: 2 and SEQ ID NO: 4, optionally as part of a vector.

Even still further provided is a method of making an antibody that bindsto sFlt-1 or a fragment thereof. The method comprises (i) expressing anisolated nucleic acid comprising a nucleotide sequence encoding theamino acid sequence of (i′) SEQ ID NO: 2, (ii′) SEQ ID NO: 4, or (iii′)SEQ ID NO: 2 and SEQ ID NO: 4 in a host cell, and (ii) isolating theantibody.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 sets forth the nucleotide (SEQ ID NO: 1) and amino acid (SEQ IDNO: 2) sequences of the variable heavy chain (VH) regions of theanti-sFlt-1 monoclonal antibody 1-833-527, in which the three heavy (H)complementarity determining regions (CDRs) are labeled as CDR-H1,CDR-H2, and CDR-H3 in the figure sequences corresponding to SEQ ID NO:1, and are underlined in the lowermost part of this figure, in thesequences corresponding to SEQ ID NO: 2.

FIG. 2 sets forth the nucleotide (SEQ ID NO: 3) and amino acid (SEQ IDNO: 4) sequences of the variable light chain (VL) regions of theanti-sFlt-1 monoclonal antibody 1-833-527, in which the three light (L)CDRs are labeled as CDR-L1, CDR-L2, and CDR-L3 in the figure sequencescorresponding to SEQ ID NO: 3, and are underlined in the lowermost partof this figure, in the sequences corresponding to SEQ ID NO: 4.

FIG. 3A-B set forth the data generated in accordance with Example 7.

FIG. 4 is a histogram showing risk stratification of Troponin I-negativepatients with sFlt-1, as described in Example 11: gray bar, sFlt-1greater than 380 pg/mL; gray bar, sFlt-1 less than or equal to 380pg/mL.

FIG. 5 is a scatter plot of sFlt-1 (ng/mL) Concentration vs. GestationalAge (weeks), as described in Example 12.

FIG. 6 is a scatter plot with linear fit of the ARCHITECT® sFlt-1 assayas described in Example 12 vs. R&D Systems sFlt-1 assay.

DETAILED DESCRIPTION

The present disclosure is predicated, at least in part, on the discoveryof a hybridoma that secretes an anti-soluble FMS-like tyrosine kinase-1(sFlt-1) antibody. The hybridoma was obtained using refined fusion,cloning and subcloning techniques as exemplified herein to obtain aclonal cell line that can grow in serum-free media earlier in the cellline discovery process. The antibody can bind to sFlt-1, in particularhuman sFlt-1, within a clinically relevant range of sFlt-1 proteinconcentration and binds to an epitope that is not bound by known,currently available anti-sFlt-1 monoclonal antibodies. The antibody canbe used in an assay, in particular an immunoassay, including thediagnosis, prognosis, and assessment of efficacy ofprophylactic/therapeutic treatment of a patient, such as a patienthaving preeclampsia or a cardiovascular disease, or for assessment ofangiogenic activity, among others. The antibody also can be used in apharmaceutical composition, such as in a method of treating a patient intherapeutic/prophylactic need of an antagonist of sFlt-1.

DEFINITIONS

The following terms are relevant to the present disclosure:

(a) “About” refers to approximately a +/−10% variation from the statedvalue. It is to be understood that such a variation is always includedin any given value provided herein, whether or not specific reference ismade to it.

(b) “Antibody” and “antibodies” refer to monoclonal antibodies,multispecific antibodies, human antibodies, humanized antibodies (fullyor partially humanized), animal antibodies (such as, but not limited to,a bird (for example, a duck or a goose), a shark, a whale, and a mammal,including a non-primate (for example, a cow, a pig, a camel, a llama, ahorse, a goat, a rabbit, a sheep, a hamster, a guinea pig, a cat, a dog,a rat, a mouse, etc.) or a non-human primate (for example, a monkey, achimpanzee, etc.), recombinant antibodies, chimeric antibodies,single-chain Fvs (“scFv”), single chain antibodies, single domainantibodies, Fab fragments, F(ab′) fragments, F(ab′)₂ fragments,disulfide-linked Fvs (“sdFv”), and anti-idiotypic (“anti-Id”)antibodies, dual-domain antibodies, dual variable domain (DVD) or triplevariable domain (TVD) antibodies (dual-variable domain immunoglobulinsand methods for making them are described in Wu, C., et al., NatureBiotechnology, 25(11):1290-1297 (2007) and PCT International ApplicationWO 2001/058956, the contents of each of which are herein incorporated byreference), and functionally active epitope-binding fragments of any ofthe above. In particular, antibodies include immunoglobulin moleculesand immunologically active fragments of immunoglobulin molecules,namely, molecules that contain an analyte-binding site. Immunoglobulinmolecules can be of any type (for example, IgG, IgE, IgM, IgD, IgA andIgY), class (for example, IgG₁, IgG₂, IgG₃, IgG₄, IgA₁ and IgA₂) orsubclass. An antibody, whose affinity (namely, K_(D), k_(d) or k_(a))has been increased or improved via the screening of a combinatoryantibody library that has been prepared using bio-display, is referredto as an “affinity maturated antibody.” For simplicity sake, an antibodyagainst an analyte is frequently referred to herein as being either an“anti-analyte antibody,” or merely an “analyte antibody” (e.g., ananti-sFlt-1 antibody or an sFlt-1 antibody).

(c) “Angiogenic activity” or “angiogenesis” refers to the formation ofnew blood vessels, which plays a role in development, as well as inwound healing, and the transition of tumors from a dormant state to amalignant one (i.e., development of cancer), among other functions.

(d) “Antibody fragment” and “antibody fragments” refer to a portion ofan intact antibody comprising the antigen-binding site or variableregion. The portion does not include the constant heavy chain domains(i.e., CH2, CH3 or CH4, depending on the antibody isotype) of the Fcregion of the intact antibody. Examples of antibody fragments include,but are not limited to, Fab fragments, Fab′ fragments, Fab′-SHfragments, F(ab′)2 fragments, Fd fragments, Fv fragments, diabodies,single-chain Fv (scFv) molecules, single-chain polypeptides containingonly one light chain variable domain, single-chain polypeptidescontaining the three CDRs of the light-chain variable domain,single-chain polypeptides containing only one heavy chain variableregion, and single-chain polypeptides containing the three CDRs of theheavy chain variable region. Such fragments are additionally describedabove under (b).

(e) “Binding Constants” are as described herein. The term “associationrate constant”, “k_(on)” or “k_(a)” as used interchangeably herein,refers to the value indicating the binding rate of an antibody to itstarget antigen or the rate of complex formation between an antibody andantigen as shown by the equation below:

Antibody (“Ab”)+Antigen (“Ag”)→Ab−Ag.

The term “dissociation rate constant”, “k_(off)” or “k_(d)” as usedinterchangeably herein, refers to the value indicating the dissociationrate of an antibody from its target antigen or separation of Ab−Agcomplex over time into free antibody and antigen as shown by theequation below:

Ab+Ag←Ab−Ag.

Methods for determining association and dissociation rate constants arewell known in the art. Using fluorescence-based techniques offers highsensitivity and the ability to examine samples in physiological buffersat equilibrium. Other experimental approaches and instruments such as aBIAcore® (biomolecular interaction analysis) assay can be used (e.g.,instrument available from BIAcore International AB, a GE Healthcarecompany, Uppsala, Sweden). Additionally, a KinExA® (Kinetic ExclusionAssay) assay, available from Sapidyne Instruments (Boise, Id.) can alsobe used.

The term “equilibrium dissociation constant” or “K_(D)” as usedinterchangeably, herein, refers to the value obtained by dividing thedissociation rate (k_(off)) by the association rate (k_(on)). Theassociation rate, the dissociation rate and the equilibrium dissociationconstant are used to represent the binding affinity of an antibody to anantigen.

(f) “Bound P1GF” refers to P1GF bound to a VEGF receptor, such as Flt-1or sFlt-1.

(g) “Bound sFlt-1” refers to sFlt-1 bound to growth factor, such as avascular endothelial growth factor (VEGF) or placental growth factor(P1GF).

(h) “Bound VEGF” refers to VEGF bound to a VEGF receptor, such as Flt-1or sFlt-1.

(i) “Cardiovascular disease” refers to various clinical diseases,disorders or conditions involving the heart, blood vessels orcirculation. The diseases, disorders or conditions can be due toatherosclerotic impairment of coronary, cerebral or peripheral arteries.Cardiovascular disease includes, but is not limited to, coronary arterydisease, peripheral vascular disease, atherosclerosis, hypertension,myocardial infarction (e.g., primary or secondary), angina pectoris,sudden cardiac death, cerebral infarction, restenosis, syncope,ischemia, transient ischemic attack, reperfusion injury, vascularocclusion, carotid obstructive disease, etc. For example, in heartfailure, “increased severity” of cardiovascular disease refers to theworsening of disease as indicated by increased NYHA classification, to,for example, Class III or Class IV, and “reduced severity” ofcardiovascular disease refers to an improvement of the disease asindicated by reduced NYHA classification, from, for example, class IIIor IV to class II or I. Cardiovascular disease also can refer to acutecoronary syndrome, and major adverse cardiac events (MACE) including butnot limited to death, myocardial infarction, or revascularization.

(j) “Component,” “components,” and “at least one component,” refergenerally to a capture antibody, a detection or conjugate antibody, acalibrator, a control, a sensitivity panel, a container, a buffer, adiluent, a salt, an enzyme, a co-factor for an enzyme, a detectionreagent, a pretreatment reagent/solution, a substrate (e.g., as asolution), a stop solution, and the like that can be included in a kitfor assay of a test sample, such as a patient urine, serum or plasmasample, in accordance with the methods described herein and othermethods known in the art. Some components can be in solution orlyophilized for reconstitution for use in an assay.

(k) “Control” refers to a composition known to not contain sFlt-1(“negative control”) or to contain sFlt-1 (“positive control”). Apositive control can comprise a known concentration of sFlt-1.“Control,” “positive control,” and “calibrator” may be usedinterchangeably herein to refer to a composition comprising a knownconcentration of sFlt-1. A “positive control” can be used to establishassay performance characteristics and is a useful indicator of theintegrity of reagents (e.g., analytes).

(l) “Eclampsia” refers to severe preeclampsia leading to the developmentof seizures. It can also include dysfunction or damage to several organsor tissues, such as the liver and central nervous system.

(m) “Epitope,” “epitopes,” or “epitopes of interest” refer to a site(s)on any molecule that is recognized and can bind to a complementarysite(s) on its specific binding partner. The molecule and specificbinding partner are part of a specific binding pair. For example, anepitope can be on a polypeptide, a protein, a hapten, a carbohydrateantigen (such as, but not limited to, glycolipids, glycoproteins orlipopolysaccharides), or a polysaccharide. Its specific binding partnercan be, but is not limited to, an antibody.

(n) “Free P1GF” refers to P1GF that is not bound to a VEGF receptor,such as Flt-1 or sFlt-1.

(o) “Free sFlt-1” refers to sFlt-1 that is not bound to growth factor.

(p) “Free VEGF” refers to VEGF that is not bound to a VEGF receptor,such as Flt-1 or sFlt-1.

(q) “Heart failure” refers to a condition in which the heart cannot pumpblood efficiently to the rest of the body. Heart failure can be due todamage to the heart or narrowing of the arteries due to infarction,cardiomyopathy (primary or secondary), hypertension, coronary arterydisease, valve disease, birth defects or infection. Heart failure canfurther be described as chronic, congestive, acute, decompensated,systolic or diastolic. The New York Heart Association (NYHA)classification describes the severity of the disease based on functionalcapacity of the patient; NYHA class can progress and/or regress based ontreatment or lack of response to treatment.

(r) “Hypertensive disorder of pregnancy (HDP)” is used herein in thecontext defined by the National Heart, Lung and Blood Institute (NHLBI)(see, e.g., Roberts et al., Hypertension 41(3): 437-445 (2003)). TheNHLBI classifies the HDP into 4 categories: (i) Preeclampsia (PE),defined as blood pressure (BP) ≧140/90 and >300 mg/24 hours proteinuriaat >20 weeks gestation; (ii) Chronic Hypertension (CHTN), defined as BP≧140/90 prior to pregnancy or <20 weeks gestation; (iii) Superimposedpreeclampsia on chronic hypertension (PE+CHTN), defined as thedevelopment of newly increased proteinuria in a woman with existingchronic hypertension >20 weeks of gestation; and (iv) GestationalHypertension (GH), defined as hypertension without proteinuria at >20weeks. Comparison of these measurements with pre-determined valuesallows the hypertensive status of the subject to be determined, forexample, to distinguish between preeclampsia and chronic hypertension.

(s) “Hypertensive status” refers to the condition of a subject withrespect to the presence or absence of a hypertensive disorder such aschronic hypertension, HDP, or a hypertensive disorder associated withanti-angiogenic drug therapy.

(t) “Identical” or “identity” as used herein in the context of two ormore polypeptide or polynucleotide sequences, can mean that thesequences have a specified percentage of residues that are the same overa specified region. The percentage can be calculated by optimallyaligning the two sequences, comparing the two sequences over thespecified region, determining the number of positions at which theidentical residue occurs in both sequences to yield the number ofmatched positions, dividing the number of matched positions by the totalnumber of positions in the specified region, and multiplying the resultby 100 to yield the percentage of sequence identity. In cases where thetwo sequences are of different lengths or the alignment produces one ormore staggered ends and the specified region of comparison includes onlya single sequence, the residues of the single sequence are included inthe denominator but not the numerator of the calculation.

(u) “Label” and “detectable label” mean a moiety attached to an antibodyor an analyte to render the reaction between the antibody and theanalyte detectable, and the antibody or analyte so labeled is referredto as “detectably labeled.” A label can produce a signal that isdetectable by visual or instrumental means. Various labels includesignal-producing substances, such as chromogens, fluorescent compounds,chemiluminescent compounds, radioactive compounds, and the like.Representative examples of labels include moieties that produce light,e.g., acridinium compounds, and moieties that produce fluorescence,e.g., fluorescein. Other labels are described herein. In this regard,the moiety, itself, may not be detectable but may become detectable uponreaction with yet another moiety. Use of the term “detectably labeled”is intended to encompass such labeling.

(v) “Linking sequence” or “linking peptide sequence” refers to a naturalor artificial polypeptide sequence that is connected to one or morepolypeptide sequences of interest (e.g., full-length, fragments, etc.).The term “connected” refers to the joining of the linking sequence tothe polypeptide sequence of interest. Such polypeptide sequences arepreferably joined by one or more peptide bonds. Linking sequences canhave a length of from about 4 to about 50 amino acids. Preferably, thelength of the linking sequence is from about 6 to about 30 amino acids.Natural linking sequences can be modified by amino acid substitutions,additions, or deletions to create artificial linking sequences.Exemplary linking sequences include, but are not limited to: (i)Histidine (His) tags, such as a 6×His tag, which has an amino acidsequence of HHHHHH (SEQ ID NO: 7), are useful as linking sequences tofacilitate the isolation and purification of polypeptides and antibodiesof interest; (ii) Enterokinase cleavage sites, like His tags, are usedin the isolation and purification of proteins and antibodies ofinterest. Often, enterokinase cleavage sites are used together with Histags in the isolation and purification of proteins and antibodies ofinterest. Various enterokinase cleavage sites are known in the art.Examples of enterokinase cleavage sites include, but are not limited to,the amino acid sequence of DDDDK (SEQ ID NO: 8) and derivatives thereof(e.g., ADDDDK (SEQ ID NO: 9), etc.); (iii) Miscellaneous sequences canbe used to link or connect the light and/or heavy chain variable regionsof single chain variable region fragments. Examples of other linkingsequences can be found in Bird et al., Science 242: 423-426 (1988);Huston et al., PNAS USA 85: 5879-5883 (1988); and McCafferty et al.,Nature 348: 552-554 (1990). Linking sequences also can be modified foradditional functions, such as attachment of drugs or attachment to solidsupports. In the context of the present disclosure, the monoclonalantibody, for example, can contain a linking sequence, such as a Histag, an enterokinase cleavage site, or both.

(w) “Patient” and “subject” may be used interchangeably herein to referto an animal, such as a bird (e.g., a duck or a goose), a shark, awhale, and a mammal, including a non-primate (for example, a cow, a pig,a camel, a llama, a horse, a goat, a rabbit, a sheep, a hamster, aguinea pig, a cat, a dog, a rat, and a mouse) and a primate (forexample, a monkey, a chimpanzee, and a human). Preferably, the patientor subject is a human, such as a non-pregnant human, a pregnant human, apost-partum human, a human at risk for preeclampsia, a human havingpreeclampsia, a human at risk for cardiovascular disease, a human havingcardiovascular disease, a human with signs and/or symptoms of an acutecoronary syndrome, or a human at risk of experiencing a major adversecardiac event (MACE).

(x) “Predetermined cutoff” and “predetermined level” refer generally toan assay cutoff value that is used to assessdiagnostic/prognostic/therapeutic efficacy results by comparing theassay results against the predetermined cutoff/level, where thepredetermined cutoff/level already has been linked or associated withvarious clinical parameters (e.g., severity of disease,progression/nonprogression/improvement, etc.). The present disclosureprovides exemplary predetermined levels. However, it is well-known thatcutoff values may vary depending on the nature of the immunoassay (e.g.,antibodies employed, etc.). It further is well within the ordinary skillof one in the art to adapt the disclosure herein for other immunoassaysto obtain immunoassay-specific cutoff values for those otherimmunoassays based on this disclosure. Whereas the precise value of thepredetermined cutoff/level may vary between assays, the correlations asdescribed herein should be generally applicable.

(y) “Preeclampsia anti-angiogenic index (PAAI)” is equal to[sFlt-1/(VEGF+P1GF)]. A PAAI of greater than 10, in particular greaterthan 20, is considered to be indicative of preeclampsia or a risk ofpreeclampsia.

(z) “Preeclampsia” refers to both a multi-system disorder, which isobserved during pregnancy and generally occurs after the 20^(th) week ofgestation, and is characterized by hypertension with or before the onsetof proteinuria and/or other signs of preeclampsia (see below), as wellas “preeclampsia-like syndrome” (PLS) associated with anti-angiogenictreatment (e.g., chemotherapy). Other signs can include edema (includingbrain and liver edema), glomerular dysfunction, and/or coagulationabnormalities. The term “preeclampsia” encompasses the NHLBI HDPdesignation of “preeclampsia/eclampsia,” as well the various clinicalforms of the disorder, including mild, moderate, and severepreeclampsia. “Preeclampsia” also includes HELLP syndrome, a variant ofsevere preeclampsia associated with hemolysis, elevated liver enzymelevels, and low platelet count.

(aa) “Preeclampsia-like syndrome (PLS)” refers to a multi-systemdisorder that is observed during anti-angiogenic treatment (e.g.,chemotherapy), which is characterized by the new onset of hypertensionwith or without proteinuria, and potentially other symptoms ofpreeclampsia (see below).

(bb) “Pretreatment reagent,” e.g., lysis, precipitation and/orsolubilization reagent, as used in a diagnostic assay as describedherein is one that lyses any cells and/or solubilizes any analyte thatis/are present in a test sample. Pretreatment is not necessary for allsamples, as described further herein. Among other things, solubilizingthe analyte (i.e., sFlt-1 or sFlt-1 fragment) entails release of theanalyte from any endogenous binding proteins present in the sample. Apretreatment reagent may be homogeneous (not requiring a separationstep) or heterogeneous (requiring a separation step). With use of aheterogeneous pretreatment reagent there is removal of any precipitatedanalyte binding proteins from the test sample prior to proceeding to thenext step of the assay. The pretreatment reagent optionally cancomprise: (a) one or more solvents and salt, (b) one or more solvents,salt and detergent, (c) detergent, (d) detergent and salt, or (e) anyreagent or combination of reagents appropriate for cell lysis and/orsolubilization of analyte.

(cc) “Quality control reagents” in the context of immunoassays and kitsdescribed herein, include, but are not limited to, calibrators,controls, and sensitivity panels. A “calibrator” or “standard” typicallyis used (e.g., one or more, such as a plurality) in order to establishcalibration (standard) curves for interpolation of the concentration ofan analyte, such as an antibody or an analyte. Alternatively, a singlecalibrator, which is near a predetermined positive/negative cutoff, canbe used. Multiple calibrators (i.e., more than one calibrator or avarying amount of calibrator(s)) can be used in conjunction so as tocomprise a “sensitivity panel.”

(dd) “Recombinant antibody” and “recombinant antibodies” refer toantibodies prepared by one or more steps, including cloning nucleic acidsequences encoding all or a part of one or more monoclonal antibodiesinto an appropriate expression vector by recombinant techniques andsubsequently expressing the antibody in an appropriate host cell. Theterms include, but are not limited to, recombinantly produced monoclonalantibodies, chimeric antibodies, humanized antibodies (fully orpartially humanized), multi-specific or multi-valent structures formedfrom antibody fragments, bifunctional antibodies, heteroconjugate Abs,DVD-Ig®s, and other antibodies as described in (i) herein.(Dual-variable domain immunoglobulins and methods for making them aredescribed in Wu, C., et al., Nature Biotechnology, 25:1290-1297 (2007)).The term “bifunctional antibody,” as used herein, refers to an antibodythat comprises a first arm having a specificity for one antigenic siteand a second arm having a specificity for a different antigenic site,i.e., the bifunctional antibodies have a dual specificity.

(ee) “Risk” refers to the possibility or probability of a particularevent occurring either presently, or, at some point in the future. “Riskstratification” or “prognosticating the risk” refers to an array ofknown clinical risk factors that allows physicians to classify patientsinto a low, moderate, high or highest risk of developing a particulardisease, disorder or condition.

(ff) “Sample,” “test sample,” and “patient sample” may be usedinterchangeably herein. The sample, such as a sample of urine, serum,plasma, amniotic fluid, cerebrospinal fluid, placental cells or tissue,endothelial cells, leukocytes, or monocytes, can be used directly asobtained from a patient or can be pre-treated, such as by filtration,distillation, extraction, concentration, centrifugation, inactivation ofinterfering components, addition of reagents, and the like, to modifythe character of the sample in some manner as discussed herein orotherwise as is known in the art.

(gg) “Series of calibrating compositions” refers to a plurality ofcompositions comprising a known concentration of sFlt-1, wherein each ofthe compositions differs from the other compositions in the series bythe concentration of sFlt-1.

(hh) “Signs and symptoms of preeclampsia” refers to both patientphysical and analytical findings (i.e., signs) and complaints (i.e.,symptoms) including hypertension (a systolic blood pressure (BP) greaterthan 140 mmHg and a diastolic BP greater than 90 mmHg after 20 weeksgestation); new onset proteinuria (1+ by dipstick on urinalysis, greaterthan 300 mg of protein in a 24-hour urine collection, or random urineprotein/creatinine ratio greater than 0.3), and resolution ofhypertension and proteinuria by 26 weeks postpartum, or upon cessationof anti-angiogenic therapy. The signs of preeclampsia can also includerenal dysfunction, glomerular endotheliosis, edema, neuropathy,coagulopathy and/or fatigue.

(ii) “Solid phase” refers to any material that is insoluble, or can bemade insoluble by a subsequent reaction. The solid phase can be chosenfor its intrinsic ability to attract and immobilize a capture agent.Alternatively, the solid phase can have affixed thereto a linking agentthat has the ability to attract and immobilize the capture agent. Thelinking agent can, for example, include a charged substance that isoppositely charged with respect to the capture agent itself or to acharged substance conjugated to the capture agent. In general, thelinking agent can be any binding partner (preferably specific) that isimmobilized on (attached to) the solid phase and that has the ability toimmobilize the capture agent through a binding reaction. The linkingagent enables the indirect binding of the capture agent to a solid phasematerial before the performance of the assay or during the performanceof the assay. The solid phase can, for example, be plastic, derivatizedplastic, magnetic or non-magnetic metal, glass or silicon, including,for example, a test tube, microtiter well, sheet, bead, microparticle,chip, and other configurations known to those of ordinary skill in theart.

(ii) “Soluble Flt-1 or sFlt-1” refers to the soluble form of the Flt-1receptor, which is also known as sVEGF-R1, is at least substantiallyidentical or identical to the protein described in GenBank Acc. No.U01134 (SEQ ID NO: 6; nucleotide sequence is SEQ ID NO: 5), and hassFlt-1 biological activity. The biological activity of an sFlt-1polypeptide can be assayed using various standard methods, e.g., byassaying sFlt-1 binding to VEGF or P1GF. sFlt-1 is used herein toinclude any sFlt-1 family member or isoform. Degradation products orfragments, such as those that result from the enzymatic cleavage of theFlt-1 receptor (for example, specific metalloproteinases released fromthe placenta can cleave the extracellular domain of the Flt-1 receptorto release the N-terminal portion of Flt-1 into circulation), areintended to be encompassed by “sFlt-1.”

(kk) “Specific binding partner” is a member of a specific binding pair.A specific binding pair comprises two different molecules, whichspecifically bind to each other through chemical or physical means.Therefore, in addition to antigen and antibody specific binding pairs ofcommon immunoassays, other specific binding pairs can include biotin andavidin (or streptavidin), carbohydrates and lectins, complementarynucleotide sequences, effector and receptor molecules, cofactors andenzymes, enzymes and enzyme inhibitors, and the like. Furthermore,specific binding pairs can include members that are analogs of theoriginal specific binding members, for example, an analyte-analog.Immunoreactive specific binding members include antigens, antigenfragments, and antibodies, including monoclonal and polyclonalantibodies as well as complexes and fragments thereof, whether isolatedor recombinantly produced.

(ll) “Specific” and “specificity” in the context of an interactionbetween members of a specific binding pair (e.g., an antigen (or afragment thereof) and an antibody (or antigenically reactive fragmentthereof)) refer to the selective reactivity of the interaction. Thephrase “specifically binds to” and analogous phrases refer to theability of antibodies (or antigenically reactive fragments thereof) tobind specifically to an antigen, such as s-Flt (or a fragment thereof),and not bind specifically to other antigens (or fragments thereof).

(mm) “Substantially identical” as used herein means that a firstsequence and a second sequence are at least from about 50% to about 99%identical over a region from about 8 to about 100 or more residues(including, in particular, any range from about 8 to about 100residues).

(nn) “Total P1GF” refers to bound P1GF and free P1GF.

(oo) “Total sFlt-1” refers to bound sFlt-1 and free sFlt-1.

(pp) “Total VEGF” refers to bound VEGF and free VEGF.

(qq) “Tracer” means an analyte or analyte fragment conjugated to alabel, such as sFlt-1 conjugated to a fluorescein moiety, wherein theanalyte conjugated to the label can effectively compete with the analytefor sites on an antibody specific for the analyte.

(rr) “Variant” as used herein means a polypeptide that differs from agiven polypeptide (i.e., anti-sFlt-1 antibody) in amino acid sequence bythe insertion, deletion, or conservative substitution of amino acids,but that retains the biological activity of the given polypeptide (i.e.,can compete with anti-sFlt-1 antibody as defined herein for binding tosFlt-1). A conservative substitution of an amino acid, i.e., replacingan amino acid with a different amino acid of similar properties (e.g.,hydrophilicity and degree and distribution of charged regions) isrecognized in the art as typically involving a minor change. These minorchanges can be identified, in part, by considering the hydropathic indexof amino acids, as understood in the art (see, e.g., Kyte et al., J.Mol. Biol. 157: 105-132 (1982)). The hydropathic index of an amino acidis based on a consideration of its hydrophobicity and charge. It isknown in the art that amino acids of similar hydropathic indexes can besubstituted and still retain protein function. In one aspect, aminoacids having hydropathic indexes of ±2 are substituted. Thehydrophilicity of amino acids also can be used to reveal substitutionsthat would result in proteins retaining biological function. Aconsideration of the hydrophilicity of amino acids in the context of apeptide permits calculation of the greatest local average hydrophilicityof that peptide, a useful measure that has been reported to correlatewell with antigenicity and immunogenicity (see, e.g., U.S. Pat. No.4,554,101, which is incorporated herein by reference). Substitution ofamino acids having similar hydrophilicity values can result in peptidesretaining biological activity, for example immunogenicity, as isunderstood in the art. In one aspect, substitutions are performed withamino acids having hydrophilicity values within ±2 of each other. Boththe hydrophobicity index and the hydrophilicity value of amino acids areinfluenced by the particular side chain of that amino acid. Consistentwith that observation, amino acid substitutions that are compatible withbiological function are understood to depend on the relative similarityof the amino acids, and particularly the side chains of those aminoacids, as revealed by the hydrophobicity, hydrophilicity, charge, size,and other properties. “Variant” also can be used to refer to anantigenically reactive fragment of an anti-sFlt-1 antibody that differsfrom the corresponding fragment of anti-sFlt-1 antibody in amino acidsequence but is still antigenically reactive and can compete with thecorresponding fragment of anti-sFlt-1 antibody for binding with sFlt-1.“Variant” also can be used to describe a polypeptide or a fragmentthereof that has been differentially processed, such as by proteolysis,phosphorylation, or other post-translational modification, yet retainsits antigen reactivity, i.e., ability to bind to sFlt-1.

The above terminology is provided for the purpose of describingparticular embodiments. The terminology is not intended to be limiting.

Anti-sFlt-1 Antibody

An isolated antibody that specifically binds to sFlt-1 or a fragmentthereof is provided. The antibody has (i) a variable heavy domain regioncomprising the amino acid sequence of SEQ ID NO: 2, (ii) a variablelight domain region comprising the amino acid sequence of SEQ ID NO: 4,or (iii) a variable heavy domain region comprising the amino acidsequence of SEQ ID NO: 2 and a variable light domain region comprisingthe amino acid sequence of SEQ ID NO: 4. The antibody binds to humansFlt-1. The antibody also binds to sFlt-1 when it is bound to a VEGF,such as P1GF. Thus, the antibody can be used to determine total sFlt-1in a test sample in accordance with the methods described herein.However, if the antibody is used in combination with an anti-sFlt-1antibody that only binds to free sFlt-1, the antibody also can be usedto determine free sFlt-1 in a test sample in accordance with the methodsdescribed herein. The antibody is preferably used as a capture antibody.The antibody can be employed with other commercially availableantibodies, e.g., clone 321 (R&D Systems catalog no. MAB321, alternatelyreferred to herein as “monoclonal antibody 321,”).

Synthetic Production

Once sequenced, polypeptides, such as a monoclonal antibody (or afragment thereof), which specifically binds to sFlt-1, can besynthesized using methods known in the art, such as, for example,exclusive solid phase synthesis, partial solid phase synthesis, fragmentcondensation, and classical solution synthesis. See, e.g., Merrifield,J. Am. Chem. Soc. 85: 2149 (1963). On solid phase, the synthesistypically begins from the C-terminal end of the peptide using analpha-amino protected resin. A suitable starting material can beprepared, for instance, by attaching the required alpha-amino acid to achloromethylated resin, a hydroxymethyl resin, or a benzhydrylamineresin. One such chloromethylated resin is sold under the tradenameBIO-BEADS SX-1 by Bio Rad Laboratories (Richmond, Calif.), and thepreparation of the hydroxymethyl resin is described by Bodonszky et al.,Chem. Ind. (London) 38: 1597 (1966). The benzhydrylamine (BHA) resin hasbeen described by Pietta and Marshall, Chem. Comm. 650 (1970) and iscommercially available from Beckman Instruments, Inc. (Palo Alto,Calif.) in the hydrochloride form. Automated peptide synthesizers arecommercially available, as are services that make peptides to order.

Thus, the polypeptides can be prepared by coupling an alpha-aminoprotected amino acid to the chloromethylated resin with the aid of, forexample, cesium bicarbonate catalyst, according to the method describedby Gisin, Helv. Chim. Acta. 56: 1467 (1973). After the initial coupling,the alpha-amino protecting group is removed by a choice of reagentsincluding trifluoroacetic acid (TFA) or hydrochloric acid (HCl)solutions in organic solvents at room temperature.

Suitable alpha-amino protecting groups include those known to be usefulin the art of stepwise synthesis of peptides. Examples of alpha-aminoprotecting groups are: acyl type protecting groups (e.g., formyl,trifluoroacetyl, and acetyl), aromatic urethane type protecting groups(e.g., benzyloxycarbonyl (Cbz) and substituted Cbz), aliphatic urethaneprotecting groups (e.g., t-butyloxycarbonyl (Boc), isopropyloxycarbonyl,and cyclohexyloxycarbonyl), and alkyl type protecting groups (e.g.,benzyl and triphenylmethyl). Boc and Fmoc are preferred protectinggroups. The side chain protecting group remains intact during couplingand is not split off during the deprotection of the amino-terminusprotecting group or during coupling. The side chain protecting groupmust be removable upon the completion of the synthesis of the finalpeptide and under reaction conditions that will not alter the targetpeptide.

After removal of the alpha-amino protecting group, the remainingprotected amino acids are coupled stepwise in the desired order. Anexcess of each protected amino acid is generally used with anappropriate carboxyl group activator such as dicyclohexylcarbodiimide(DCC) in solution, for example, in methylene chloride and dimethylformamide (DMF) mixtures.

After the desired amino acid sequence has been completed, the desiredpeptide is decoupled from the resin support by treatment with a reagent,such as TFA or hydrogen fluoride (HF), which not only cleaves thepeptide from the resin, but also cleaves all remaining side chainprotecting groups. When the chloromethylated resin is used, HF treatmentresults in the formation of the free peptide acids. When thebenzhydrylamine resin is used, HF treatment results directly in the freepeptide amide. Alternatively, when the chloromethylated resin isemployed, the side chain protected peptide can be decoupled by treatmentof the peptide resin with ammonia to give the desired side chainprotected amide or with an alkylamine to give a side chain protectedalkylamide or dialkylamide. Side chain protection is then removed in theusual fashion by treatment with hydrogen fluoride to give the freeamides, alkylamides, or dialkylamides.

These and other solid phase peptide synthesis procedures are well-knownin the art. Such procedures are also described by Stewart and Young inSolid Phase Peptide Syntheses (2nd Ed., Pierce Chemical Company, 1984).

Recombinant Production

A polypeptide, such as a monoclonal antibody (or a fragment thereof),which specifically binds to sFlt-1, can be recombinantly produced usingmethods known in the art. For example, an isolated nucleic acidcomprising a nucleotide sequence encoding the antibody (or a fragmentthereof) can be expressed in a host cell, and the antibody can beisolated. The isolated nucleic acid can comprise a nucleotide sequenceencoding the amino acid sequence of SEQ ID NO: 2 (VH domain region),such as the nucleotide sequence of SEQ ID NO: 1, and/or a nucleotidesequence encoding the amino acid sequence of SEQ ID NO: 4 (VL domainregion), such as the nucleotide sequence of SEQ ID NO: 3. The isolatednucleic acid can be synthesized with an oligonucleotide synthesizer, forexample. One of ordinary skill in the art will readily appreciate that,due to the degeneracy of the genetic code, more than one nucleotidesequence can encode a given amino acid sequence. In this regard, anucleotide sequence encoding an amino acid sequence that issubstantially identical to SEQ ID NO: 2 and/or an amino acid sequencethat is substantially identical to SEQ ID NO: 4 can be used, providedthat the variant antibody as expressed competes with the antibodycomprising the amino acid sequence of SEQ ID NO: 2 and/or the amino acidsequence of SEQ ID NO: 4 for the same epitope on sFlt-1. Codons, whichare favored by a given host cell, preferably are selected forrecombinant production. A nucleotide sequence encoding the amino acidsequence of SEQ ID NO: 2 and/or a nucleotide sequence encoding the aminoacid sequence of SEQ ID NO: 4 can be combined with other nucleotidesequences using polymerase chain reaction (PCR), ligation, or ligationchain reaction (LCR) to encode an anti-sFlt-1 antibody or antigenicallyreactive fragment thereof. The individual oligonucleotides typicallycontain 5′ or 3′ overhangs for complementary assembly. Once assembled,the nucleotide sequence encoding an anti-sFlt-1 antibody orantigenically reactive fragment thereof can be inserted into a vector,operably linked to control sequences as necessary for expression in agiven host cell, and introduced (such as by transformation ortransfection) into a host cell. The nucleotide sequence can be furthermanipulated (for example, linked to one or more nucleotide sequencesencoding additional immunoglobulin domains, such as additional constantregions) and/or expressed in a host cell.

Although not all vectors and expression control sequences may functionequally well to express a polynucleotide sequence of interest and notall hosts function equally well with the same expression system, it isbelieved that those skilled in the art will be able to make a selectionamong these vectors, expression control sequences, optimized codons, andhosts for use in the present disclosure without any undueexperimentation. For example, in selecting a vector, the host must beconsidered because the vector must be able to replicate in it or be ableto integrate into the chromosome. The vector's copy number, the abilityto control that copy number, and the expression of any other proteinsencoded by the vector, such as antibiotic markers, should also beconsidered. In selecting an expression control sequence, a variety offactors also can be considered. These include, but are not limited to,the relative strength of the sequence, its controllability, and itscompatibility with the nucleotide sequence encoding the anti-sFlt-1antibody, particularly with regard to potential secondary structures.Hosts should be selected by consideration of their compatibility withthe chosen vector, their codon usage, their secretion characteristics,their ability to fold the polypeptide correctly, their fermentation orculture requirements, their ability (or lack thereof) to glycosylate theprotein, and the ease of purification of the products encoded by thenucleotide sequence, etc.

The recombinant vector can be an autonomously replicating vector,namely, a vector existing as an extrachromosomal entity, the replicationof which is independent of chromosomal replication (such as a plasmid).Alternatively, the vector can be one which, when introduced into a hostcell, is integrated into the host cell genome and replicated togetherwith the chromosome(s) into which it has been integrated.

The vector is preferably an expression vector in which thepolynucleotide sequence encoding the anti-sFlt-1 antibody is operablylinked to additional segments required for transcription of thepolynucleotide sequence. The vector is typically derived from plasmid orviral DNA. A number of suitable expression vectors for expression in thehost cells mentioned herein are commercially available or described inthe literature. Useful expression vectors for eukaryotic hosts, include,but are not limited to, vectors comprising expression control sequencesfrom SV40, bovine papilloma virus, adenovirus and cytomegalovirus.Specific vectors include pcDNA3.1 (+)\Hyg (Invitrogen Corp., Carlsbad,Calif.) and pCI-neo (Stratagene, La Jolla, Calif.). Examples ofexpression vectors for use in yeast cells include, but are not limitedto, the 2μ plasmid and derivatives thereof, the POT1 vector (see, e.g.,U.S. Pat. No. 4,931,373), the pJSO37 vector (described in Okkels, Ann.New York Acad. Sci. 782: 202-207 (1996)) and pPICZ A, B or C(Invitrogen). Examples of expression vectors for use in insect cellsinclude, but are not limited to, pVL941, pBG311 (Cate et al., Cell 45:685-698 (1986)), and pBluebac 4.5 and pMelbac (both of which areavailable from Invitrogen).

Other vectors that can be used allow the nucleotide sequence encodingthe anti-sFlt-1 antibody to be amplified in copy number. Suchamplifiable vectors are well-known in the art. These vectors include,but are not limited to, those vectors that can be amplified bydihydrofolate reductase (DHFR) amplification (see, for example,Kaufinan, U.S. Pat. No. 4,470,461; and Kaufman et al., Mol. Cell. Biol.2: 1304-1319 (1982)) and glutamine synthetase (GS) amplification (see,for example, U.S. Pat. No. 5,122,464 and European Pat. App. Pub. No. 0338 841).

The recombinant vector can further comprise a nucleotide sequenceenabling the vector to replicate in the host cell in question. Anexample of such a sequence for use in a mammalian host cell is the SV40origin of replication. Suitable sequences enabling the vector toreplicate in a yeast cell are the yeast plasmid 2μ replication genes REP1-3 and origin of replication.

The vector can also comprise a selectable marker, namely, a gene orpolynucleotide, the product of which complements a defect in the hostcell, such as the gene coding for DHFR or the Schizosaccharomyces pombeTPI gene (see, e.g., Russell, Gene 40: 125-130 (1985)), or one whichconfers resistance to a drug, such as ampicillin, kanamycin,tetracycline, chloramphenicol, neomycin, hygromycin or methotrexate. Forfilamentous fungi, selectable markers include, but are not limited to,amdS, pyrG, arcB, niaD and sC.

Also present in the vector are “control sequences,” which are anycomponents that are necessary or advantageous for the expression of theanti-sFlt-1 antibody. Each control sequence can be native or foreign tothe nucleotide sequence encoding the anti-sFlt-1 antibody. Such controlsequences include, but are not limited to, a leader, a polyadenylationsequence, a propeptide sequence, a promoter, an enhancer or an upstreamactivating sequence, a signal peptide sequence, and a transcriptionterminator. At a minimum, the control sequences include at least onepromoter operably linked to the polynucleotide sequence encoding theanti-sFlt-1 antibody.

By “operably linked” is meant the covalent joining of two or morenucleotide sequences, by means of enzymatic ligation or otherwise, in aconfiguration relative to one another such that the normal function ofthe sequences can be performed. For example, a nucleotide sequenceencoding a presequence or secretory leader is operably linked to anucleotide sequence for a polypeptide if it is expressed as a preproteinthat participates in the secretion of the polypeptide; a promoter orenhancer is operably linked to a coding sequence if it affects thetranscription of the sequence; a ribosome binding site is operablylinked to a coding sequence if it is positioned so as to facilitatetranslation. Generally, “operably linked” means that the nucleotidesequences being linked are contiguous and, in the case of a secretoryleader, contiguous and in the same reading frame. Linking isaccomplished by ligation at convenient restriction sites. If such sitesdo not exist, then synthetic oligonucleotide adaptors or linkers can beused, in conjunction with standard recombinant DNA methods.

A wide variety of expression control sequences can be used in thecontext of the present disclosure. Such useful expression controlsequences include the expression control sequences associated withstructural genes of the foregoing expression vectors as well as anysequence known to control the expression of genes of prokaryotic oreukaryotic cells or their viruses, and various combinations thereof.Examples of suitable control sequences for directing transcription inmammalian cells include the early and late promoters of SV40 andadenovirus, for example, the adenovirus 2 major late promoter, the MT-1(metallothionein gene) promoter, the human cytomegalovirusimmediate-early gene promoter (CMV), the human elongation factor 1α(EF-1α) promoter, the Drosophila minimal heat shock protein 70 promoter,the Rous Sarcoma Virus (RSV) promoter, the human ubiquitin C (UbC)promoter, the human growth hormone terminator, SV40 or adenovirus E1bregion polyadenylation signals and the Kozak consensus sequence (Kozak,J. Mol. Biol. 196: 947-50 (1987)).

In order to improve expression in mammalian cells a synthetic intron canbe inserted in the 5′ untranslated region of a polynucleotide sequenceencoding the antibody or a fragment thereof. An example of a syntheticintron is the synthetic intron from the plasmid pCI-Neo (available fromPromega Corporation, Madison, Wis.).

Examples of suitable control sequences for directing transcription ininsect cells include, but are not limited to, the polyhedrin promoter,the P10 promoter, the baculovirus immediate early gene 1 promoter, thebaculovirus 39K delayed-early gene promoter, and the SV40polyadenylation sequence.

Examples of suitable control sequences for use in yeast host cellsinclude the promoters of the yeast α-mating system, the yeast triosephosphate isomerase (TPI) promoter, promoters from yeast glycolyticgenes or alcohol dehydrogenase genes, the ADH2-4-c promoter and theinducible GAL promoter.

Examples of suitable control sequences for use in filamentous fungalhost cells include the ADH3 promoter and terminator, a promoter derivedfrom the genes encoding Aspergillus oryzae TAKA amylase triose phosphateisomerase or alkaline protease, an A. niger α-amylase, A. niger or A.nidulas glucoamylase, A. nidulans acetamidase, Rhizomucor mieheiaspartic proteinase or lipase, the TPI1 terminator, and the ADH3terminator.

The polynucleotide sequence encoding the antibody of interest may or maynot also include a polynucleotide sequence that encodes a signalpeptide. The signal peptide is present when the anti-sFlt-1 antibody isto be secreted from the cells in which it is expressed. Such signalpeptide, if present, should be one recognized by the cell chosen forexpression of the polypeptide. The signal peptide can be homologous orheterologous to the anti-sFlt-1 monoclonal antibody or can be homologousor heterologous to the host cell, i.e., a signal peptide normallyexpressed from the host cell or one which is not normally expressed fromthe host cell. Accordingly, the signal peptide can be prokaryotic, forexample, derived from a bacterium, or eukaryotic, for example, derivedfrom a mammalian, insect, filamentous fungal, or yeast cell.

The presence or absence of a signal peptide will, for example, depend onthe expression host cell used for the production of the anti-sFlt-1antibody. For use in filamentous fungi, the signal peptide canconveniently be derived from a gene encoding an Aspergillus sp. amylaseor glucoamylase, a gene encoding a Rhizomucor miehei lipase or proteaseor a Humicola lanuginosa lipase. For use in insect cells, the signalpeptide can be derived from an insect gene (see, e.g., Int'l Pat. App.Pub. No. WO 90/05783), such as the lepidopteran Manduca sextaadipokinetic hormone precursor (see, e.g., U.S. Pat. No. 5,023,328), thehoneybee melittin (Invitrogen), ecdysteroid UDP glucosyltransferase(egt) (Murphy et al., Protein Expression and Purification 4: 349-357(1993), or human pancreatic lipase (hpl) (Methods in Enzymology 284:262-272 (1997)).

Specific examples of signal peptides for use in mammalian cells includemurine Ig kappa light chain signal peptide (Coloma, J. Imm. Methods 152:89-104 (1992)). Suitable signal peptides for use in yeast cells includethe α-factor signal peptide from S. cerevisiae (see, e.g., U.S. Pat. No.4,870,008), the signal peptide of mouse salivary amylase (see, e.g.,Hagenbuchle et al., Nature 289: 643-646 (1981)), a modifiedcarboxypeptidase signal peptide (see, e.g., Valls et al., Cell 48:887-897 (1987)), the yeast BAR1 signal peptide (see, e.g., Int'l Pat.App. Pub. No. WO 87/02670), and the yeast aspartic protease 3 (YAP3)signal peptide (see, e.g., Egel-Mitani et al., Yeast 6: 127-137 (1990)).

Any suitable host can be used to produce the anti-sFlt-1 antibody,including bacteria, fungi (including yeasts), plant, insect, mammal orother appropriate animal cells or cell lines, as well as transgenicanimals or plants. Examples of bacterial host cells include, but are notlimited to, gram-positive bacteria, such as strains of Bacillus, forexample, B. brevis or B. subtilis, Pseudomonas or Streptomyces, orgram-negative bacteria, such as strains of E. coli. The introduction ofa vector into a bacterial host cell can, for instance, be effected byprotoplast transformation (see, for example, Chang et al., Molec. Gen.Genet. 168: 111-115 (1979)), using competent cells (see, for example,Young et al., J. of Bacteriology 81: 823-829 (1961), or Dubnau et al.,J. of Molec. Biol. 56: 209-221 (1971)), electroporation (see, forexample, Shigekawa et al., Biotechniques 6: 742-751 (1988)), orconjugation (see, for example, Koehler et al., J. of Bacteriology 169:5771-5278 (1987)).

Examples of suitable filamentous fungal host cells include, but are notlimited to, strains of Aspergillus, for example, A. oryzae, A. niger, orA. nidulans, Fusarium or Trichoderma. Fungal cells can be transformed bya process involving protoplast formation, transformation of theprotoplasts, and regeneration of the cell wall using techniques known tothose ordinarily skilled in the art. Suitable procedures fortransformation of Aspergillus host cells are described in European Pat.App. Pub. No. 238 023 and U.S. Pat. No. 5,679,543. Suitable methods fortransforming Fusarium species are described by Malardier et al., Gene78: 147-156 (1989), and Int'l Pat. App. Pub. No. WO 96/00787. Yeast canbe transformed using the procedures described by Becker and Guarente, InAbelson, J. N. and Simon, M. I., editors, Guide to Yeast Genetics andMolecular Biology, Methods in Enzymology 194: 182-187, Academic Press,Inc., New York; Ito et al, J. of Bacteriology 153: 163 (1983); andHinnen et al., PNAS USA 75: 1920 (1978).

Examples of suitable yeast host cells include strains of Saccharomyces,for example, S. cerevisiae, Schizosaccharomyces, Klyveromyces, Pichia,such as P. pastoris or P. methanolica, Hansenula, such as H. polymorphaor yarrowia. Methods for transforming yeast cells with heterologouspolynucleotides and producing heterologous polypeptides therefrom aredisclosed by Clontech Laboratories, Inc, Palo Alto, Calif., USA (in theproduct protocol for the Yeastmaker™ Yeast Tranformation System Kit),and by Reeves et al., FEMS Microbiology Letters 99: 193-198 (1992),Manivasakam et al., Nucleic Acids Research 21: 4414-4415 (1993), andGaneva et al., FEMS Microbiology Letters 121: 159-164 (1994).

Examples of suitable insect host cells include, but are not limited to,a Lepidoptora cell line, such as Spodoptera frugiperda (Sf9 or Sf21) orTrichoplusia ni cells (High Five) (see, e.g., U.S. Pat. No. 5,077,214).Transformation of insect cells and production of heterologouspolypeptides are well-known to those skilled in the art.

Examples of suitable mammalian host cells include Chinese hamster ovary(CHO) cell lines, simian (e.g., Green Monkey) cell lines (COS), mousecells (for example, NS/O), baby hamster kidney (BHK) cell lines, humancells (such as human embryonic kidney (HEK) cells (e.g., HEK 293 cells(A.T.C.C. Accession No. CRL-1573))), myeloma cells that do not otherwiseproduce immunoglobulin protein, and plant cells in tissue culture.Preferably, the mammalian host cells are CHO cell lines and HEK 293 celllines. Another preferred host cell is the B3.2 cell line (e.g., AbbottLaboratories, Abbott Bioresearch Center), or another dihydrofolatereductase deficient (DHFR⁻) CHO cell line (e.g., available fromInvitrogen).

Methods for introducing exogenous polynucleotides into mammalian hostcells include calcium phosphate-mediated transfection, electroporation,DEAE-dextran mediated transfection, liposome-mediated transfection,viral vectors and the transfection method described by Life TechnologiesLtd, Paisley, UK using Lipofectamine™ 2000. These methods are well-knownin the art and are described, for example, by Ausbel et al. (eds.),Current Protocols in Molecular Biology, John Wiley & Sons, New York, USA(1996). The cultivation of mammalian cells is conducted according toestablished methods, e.g., as disclosed in Jenkins, Ed., Animal CellBiotechnology, Methods and Protocols, Human Press Inc. Totowa, N.J., USA(1999), and Harrison and Rae, General Techniques of Cell Culture,Cambridge University Press (1997).

In the production methods, cells are cultivated in a nutrient mediumsuitable for production of the anti-sFlt-1 antibody using methods knownin the art. For example, cells are cultivated by shake flaskcultivation, small-scale or large-scale fermentation (includingcontinuous, batch, fed-batch, or solid state fermentations) inlaboratory or industrial fermenters performed in a suitable medium andunder conditions allowing the anti-human sFlt-1 monoclonal antibody tobe expressed and/or isolated. The cultivation takes place in a suitablenutrient medium comprising carbon and nitrogen sources and inorganicsalts, using procedures known in the art. Suitable media are availablefrom commercial suppliers or can be prepared according to publishedcompositions (e.g., in catalogues of the American Type CultureCollection). If the anti-sFlt-1 antibody is secreted into the nutrientmedium, it can be recovered directly from the medium. If the anti-sFlt-1antibody is not secreted, it can be recovered from cell lysates.

The resulting anti-sFlt-1 antibody can be recovered by methods known inthe art. For example, the anti-sFlt-1 antibody can be recovered from thenutrient medium by conventional procedures including, but not limitedto, centrifugation, filtration, extraction, spray drying, evaporation,or precipitation.

The anti-sFlt-1 antibody can be purified by a variety of proceduresknown in the art including, but not limited to, chromatography (such as,but not limited to, ion exchange, affinity, hydrophobic,chromatofocusing, and size exclusion), electrophoretic procedures (suchas, but not limited to, preparative isoelectric focusing), differentialsolubility (such as, but not limited to, ammonium sulfateprecipitation), SDS-PAGE, or extraction (see, for example, Janson andRyden, editors, Protein Purification, VCH Publishers, New York (1989)).

Antibody fragments are also contemplated. For example, the antibodyfragment can include, but is not limited to, a Fab, a Fab′, a Fab′-SHfragment, a di-sulfide linked Fv, a single chain Fv (scFv) and a F(ab′)₂fragment. Various techniques are known to those skilled in the art forthe production of antibody fragments. For example, such fragments can bederived via proteolytic digestion of intact antibodies (see, forexample, Morimoto et al., J. Biochem. Biophys. Methods 24: 107-117(1992), and Brennan et al., Science 229: 81 (1985)) or produced directlyby recombinant host cells. For example, Fab′-SH fragments can bedirectly recovered from E. coli and chemically coupled to form F(ab′)₂fragments (see, e.g., Carter et al., Bio/Technology 10: 163-167 (1992)).In another embodiment, the F(ab′)₂ is formed using the leucine zipperGCN4 to promote assembly of the F(ab′)₂ molecule. Alternatively, Fv, Fabor F(ab′)₂ fragments can be isolated directly from recombinant host cellculture. Single chain variable region fragments (scFv) are made bylinking light and/or heavy chain variable regions by using a shortlinking peptide or sequence (see, e.g., Bird et al., Science 242:423-426 (1998)). The single chain variants can be produced eitherrecombinantly or synthetically. For synthetic production of scFv, anautomated synthesizer can be used. For recombinant production of scFv, asuitable plasmid containing polynucleotide that encodes the scFv can beintroduced into a suitable host cell, either eukaryotic, such as yeast,plant, insect or mammalian cells, or prokaryotic, such as E. coli.Polynucleotides encoding the scFv of interest can be made by routinemanipulations such as ligation of polynucleotides. The resultant scFvcan be isolated using standard protein purification techniques known inthe art. Moreover, other forms of single-chain antibodies, such asdiabodies are also contemplated by the present disclosure. Diabodies arebivalent, bispecific antibodies in which VH and VL domains are expressedon a single polypeptide chain, but using a linker that is too short toallow for pairing between the two domains on the same chain, therebyforcing the domains to pair with complementary domains of another chainand creating two antigen-binding sites (see, for example, Holliger etal., PNAS USA 90: 6444-6448 (1993); and Poljak et al., Structure 2:1121-1123 (1994)).

The antibody and antigenically reactive fragment thereof have a varietyof uses. In one aspect, the antibody (or a fragment thereof) can be usedas one or more immunodiagnostic reagents. For example, the antibodies ofthe present disclosure can be used as one or more immunodiagnosticreagents in one or more methods for detecting the presence of sFlt-1 ina test sample. More specifically, the antibody (or antigenicallyreactive fragment thereof) can be used as a capture antibody or adetection antibody in an immunoassay to detect the presence of sFlt-1,such as human sFlt-1 (or a fragment thereof), in a test sample.

Antibody Production

Other antibodies (or fragments thereof) that specifically bind to sFlt-1(or a fragment thereof) can be made using a variety of differenttechniques known in the art. For example, polyclonal and monoclonalantibodies can be raised by immunizing a suitable subject (such as, butnot limited to, a rabbit, a goat, a mouse, or other mammal) with animmunogenic preparation, which contains a suitable immunogen. Theimmunogen can be enriched/purified and isolated from a cell thatproduces it using affinity chromatography, immune-precipitation or othertechniques, which are well-known in the art. Alternatively, immunogencan be prepared using chemical synthesis using routine techniques knownin the art (such as, but not limited to, a synthesizer). The antibodiesraised in the subject can then be screened to determine if theantibodies bind to the immunogen (or a fragment thereof).

The unit dose of immunogen (namely, the purified protein, tumor cellexpressing the protein, or recombinantly expressed immunogen (or afragment or a variant (or a fragment thereof) thereof) and theimmunization regimen will depend upon the subject to be immunized, itsimmune status, and the body weight of the subject. To enhance an immuneresponse in the subject, an immunogen can be administered with anadjuvant, such as Freund's complete or incomplete adjuvant.

Immunization of a subject with an immunogen as described above induces apolyclonal antibody response. The antibody titer in the immunizedsubject can be monitored over time by standard techniques such as anELISA using an immobilized antigen.

Other methods of raising antibodies include using transgenic mice, whichexpress human immunoglobin genes (see, for example, Int'l Pat. App. Pub.Nos. WO 91/00906, WO 91/10741, and WO 92/03918). Alternatively, humanmonoclonal antibodies can be produced by introducing an antigen intoimmune-deficient mice that have been engrafted with humanantibody-producing cells or tissues (for example, human bone marrowcells, peripheral blood lymphocytes (PBL), human fetal lymph nodetissue, or hematopoietic stem cells). Such methods include raisingantibodies in SCID-hu mice (see, for example, Int'l Pat. App. Pub. No.WO 93/05796; U.S. Pat. No. 5,411,749; or McCune et al., Science 241:1632-1639 (1988)) or Rag-1/Rag-2 deficient mice. Human antibody-immunedeficient mice are also commercially available. For example, Rag-2deficient mice are available from Taconic Farms (Germantown, N.Y.).

Monoclonal antibodies can be generated by immunizing a subject with animmunogen. At the appropriate time after immunization, for example, whenthe antibody titers are at a sufficiently high level, antibody-producingcells can be harvested from an immunized animal and used to preparemonoclonal antibodies using standard techniques. For example, theantibody-producing cells can be fused by standard somatic cell fusionprocedures with immortalizing cells, such as myeloma cells, to yieldhybridoma cells. Such techniques are well-known in the art, and include,for example, the hybridoma technique as originally developed by Kohlerand Milstein, Nature 256: 495-497 (1975)), the human B cell hybridomatechnique (Kozbar et al., Immunology Today 4: 72 (1983)), and theEpstein-Barr virus (EBV)-hybridoma technique to produce human monoclonalantibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, AlanR. Liss, Inc. pp. 77-96 (1985)). The technology for producing monoclonalantibody hybridomas is well-known to those skilled in the art.

Monoclonal antibodies also can be made by harvesting antibody-producingcells, for example, splenocytes, from transgenic mice, which expresshuman immunoglobulin genes and which have been immunized with theimmunogen. The splenocytes can be immortalized through fusion with humanmyelomas or through transformation with EBV. These hybridomas can bemade using human B cell- or EBV-hybridoma techniques described in theart (See, for example, Boyle et al., European Pat. Pub. No. 0 614 984).

Hybridoma cells producing a monoclonal antibody, which specificallybinds to the immunogen, are detected by screening the hybridoma culturesupernatants by, for example, screening to select antibodies thatspecifically bind to the immobilized immunogen (or a fragment thereof),or by testing the antibodies as described herein to determine if theantibodies have the desired characteristics, namely, the ability to bindto immunogen (or a fragment thereof). After hybridoma cells areidentified that produce antibodies of the desired specificity, theclones may be subcloned, e.g., by limiting dilution procedures, forexample the procedure described by Wands et al. (Gastroenterology 80:225-232 (1981)), and grown by standard methods.

Hybridoma cells that produce monoclonal antibodies that test positive inthe screening assays described herein can be cultured in a nutrientmedium under conditions and for a time sufficient to allow the hybridomacells to secrete the monoclonal antibodies into the culture medium, tothereby produce whole antibodies. Tissue culture techniques and culturemedia suitable for hybridoma cells are generally described in the art(See, for example, R. H. Kenneth, in Monoclonal Antibodies: A NewDimension In Biological Analyses, Plenum Publishing Corp., New York,N.Y. (1980)). Conditioned hybridoma culture supernatant containing theantibody can then be collected. The monoclonal antibodies secreted bythe subclones optionally can be isolated from the culture medium byconventional immunoglobulin purification procedures such as, forexample, protein A chromatography, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

Monoclonal antibodies can be engineered by constructing a recombinantcombinatorial immunoglobulin library and screening the library with theimmunogen or a fragment thereof. Kits for generating and screening phagedisplay libraries are commercially available (See, for example, thePharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01; andthe Stratagene SurfZAP Phage Display Kit, Catalog No. 240612). Likewise,yeast display vectors are known in the art and are commerciallyavailable (for example, pYD1 available from Invitrogen). Briefly, theantibody library is screened to identify and isolate phages or yeastcells that express an antibody that specifically binds to the immunogenor a fragment thereof. Preferably, the primary screening of the libraryinvolves screening with an immobilized immunogen or a fragment thereof.

Following screening, the display phage or yeast is isolated and thepolynucleotide encoding the selected antibody can be recovered from thedisplay phage or yeast (for example, from the phage or yeast genome) andsubcloned into other expression vectors (e.g., into Saccharomycescerevesiae cells, for example EBY100 cells (Invitrogen)) by well-knownrecombinant DNA techniques. The polynucleotide can be furthermanipulated (for example, linked to nucleic acid encoding additionalimmunoglobulin domains, such as additional constant regions) and/orexpressed in a host cell.

Once a monoclonal antibody that specifically binds to sFlt-1 is obtainedin accordance with methods described above, it can be sequenced inaccordance with methods known in the art. The antibody then can be madeusing recombinant DNA technology, chemical synthesis, or a combinationof chemical synthesis and recombinant DNA technology as described above.

Furthermore, in some aspects of the disclosure, it may be possible toemploy commercially available anti-sFlt-1 antibodies or methods forproduction of anti-sFlt-1 antibodies as described in the literature.These include, but are not limited to, those available from Santa CruzBiotechnology, Inc. (Santa Cruz, Calif.) and R&D Systems (Minneapolis,Minn.).

Kit

A kit for assaying a test sample for sFlt-1 (or a fragment thereof) isalso provided. The kit comprises at least one component for assaying thetest sample for sFlt-1 and instructions for assaying the test sample forsFlt-1 (or a fragment thereof). The at least one component includes atleast one composition comprising an isolated antibody that specificallybinds to sFlt-1 (or a fragment thereof). The antibody has (i) a variableheavy domain region comprising the amino acid sequence of SEQ ID NO: 2,(ii) a variable light domain region comprising the amino acid sequenceof SEQ ID NO: 4, or (iii) a variable heavy domain region comprising theamino acid sequence of SEQ ID NO: 2 and a variable light domain regioncomprising the amino acid sequence of SEQ ID NO: 4. The antibody isoptionally detectably labeled.

For example, the kit can comprise instructions for assaying the testsample for sFlt-1 (or a fragment thereof) by immunoassay, e.g.,chemiluminescent microparticle immunoassay. The instructions can be inpaper form or computer-readable form, such as a disk, CD, DVD, or thelike. The antibody can be an sFlt-1 capture antibody and/or an sFlt-1detection antibody. Alternatively or additionally, the kit can comprisea calibrator or control, e.g., purified, and optionally lyophilized,sFlt-1 (or a fragment thereof), and/or at least one container (e.g.,tube, microtiter plates or strips, which can be already coated with ananti-sFlt-1 monoclonal antibody) for conducting the assay, and/or abuffer, such as an assay buffer or a wash buffer, either one of whichcan be provided as a concentrated solution, a substrate solution for thedetectable label (e.g., an enzymatic label), or a stop solution.Preferably, the kit comprises all components, i.e., reagents, standards,buffers, diluents, etc., which are necessary to perform the assay. Theinstructions also can include instructions for generating a standardcurve or a reference standard for purposes of quantifying sFlt-1.

Any antibodies, which are provided in the kit, such as recombinantantibodies specific for sFlt-1, can incorporate a detectable label, suchas a fluorophore, radioactive moiety, enzyme, biotin/avidin label,chromophore, chemiluminescent label, or the like, or the kit can includereagents for labeling the antibodies or reagents for detecting theantibodies (e.g., detection antibodies) and/or for labeling the analytesor reagents for detecting the analyte. The antibodies, calibratorsand/or controls can be provided in separate containers or pre-dispensedinto an appropriate assay format, for example, into microtiter plates.

Optionally, the kit includes quality control components (for example,sensitivity panels, calibrators, and positive controls). Preparation ofquality control reagents is well-known in the art and is described oninsert sheets for a variety of immunodiagnostic products. Sensitivitypanel members optionally are used to establish assay performancecharacteristics, and further optionally are useful indicators of theintegrity of the immunoassay kit reagents, and the standardization ofassays.

The kit can also optionally include other reagents required to conduct adiagnostic assay or facilitate quality control evaluations, such asbuffers, salts, enzymes, enzyme co-factors, substrates, detectionreagents, and the like. Other components, such as buffers and solutionsfor the isolation and/or treatment of a test sample (e.g., pretreatmentreagents), also can be included in the kit. The kit can additionallyinclude one or more other controls. One or more of the components of thekit can be lyophilized, in which case the kit can further comprisereagents suitable for the reconstitution of the lyophilized components.

The various components of the kit optionally are provided in suitablecontainers as necessary, e.g., a microtiter plate. The kit can furtherinclude containers for holding or storing a sample (e.g., a container orcartridge for a urine sample). Where appropriate, the kit optionallyalso can contain reaction vessels, mixing vessels, and other componentsthat facilitate the preparation of reagents or the test sample. The kitcan also include one or more instrument for assisting with obtaining atest sample, such as a syringe, pipette, forceps, measured spoon, or thelike.

If the detectable label is at least one acridinium compound, the kit cancomprise at least one acridinium-9-carboxamide, at least oneacridinium-9-carboxylate aryl ester, or any combination thereof. If thedetectable label is at least one acridinium compound, the kit also cancomprise a source of hydrogen peroxide, such as a buffer, solution,and/or at least one basic solution. If desired, the kit can contain asolid phase, such as a magnetic particle, bead, test tube, microtiterplate, cuvette, membrane, scaffolding molecule, film, filter paper, discor chip.

Method of Determining the Presence, Amount or Concentration of sFlt-1(or a Fragment Thereof) in a Test Sample

The present disclosure provides a method for determining the presence,amount or concentration of sFlt-1 (or a fragment thereof) in a testsample. Any suitable assay as is known in the art can be used in themethod. Examples include, but are not limited to, immunoassay, such assandwich immunoassay (e.g., monoclonal-polyclonal sandwich immunoassays,including radioisotope detection (radioimmunoassay (RIA)) and enzymedetection (enzyme immunoassay (EIA) or enzyme-linked immunosorbent assay(ELISA) (e.g., Quantikine ELISA assays, R&D Systems, Minneapolis,Minn.)), competitive inhibition immunoassay (e.g., forward and reverse),fluorescence polarization immunoassay (FPIA), enzyme multipliedimmunoassay technique (EMIT), bioluminescence resonance energy transfer(BRET), and homogeneous chemiluminescent assay, etc. In a SELDI-basedimmunoassay, a capture reagent that specifically binds sFlt-1 (or afragment thereof) of interest is attached to the surface of a massspectrometry probe, such as a pre-activated protein chip array. ThesFlt-1 (or a fragment thereof) is then specifically captured on thebiochip, and the captured sFlt-1 (or a fragment thereof) is detected bymass spectrometry. Alternatively, the sFlt-1 (or a fragment thereof) canbe eluted from the capture reagent and detected by traditional MALDI(matrix-assisted laser desorption/ionization) or by SELDI. Achemiluminescent microparticle immunoassay, in particular one employingthe ARCHITECT® automated analyzer (Abbott Laboratories, Abbott Park,Ill.), is an example of a preferred immunoassay.

Methods well-known in the art for collecting, handling and processingurine, blood, serum and plasma, and other body fluids, are used in thepractice of the present disclosure, for instance, when the antibodiesaccording to the present disclosure are employed as immunodiagnosticreagents, and/or in an sFlt-1 immunoassay kit. The test sample cancomprise further moieties in addition to the sFlt-1 analyte of interest,such as antibodies, antigens, haptens, hormones, drugs, enzymes,receptors, proteins, peptides, polypeptides, oligonucleotides orpolynucleotides. For example, the sample can be a whole blood sampleobtained from a subject. It can be necessary or desired that a testsample, particularly whole blood, be treated prior to immunoassay asdescribed herein, e.g., with a pretreatment reagent. Even in cases wherepretreatment is not necessary (e.g., most urine samples), pretreatmentoptionally can be done for mere convenience (e.g., as part of a regimenon a commercial platform).

The pretreatment reagent can be any reagent appropriate for use with theimmunoassay and kits of the invention. The pretreatment optionallycomprises: (a) one or more solvents (e.g., methanol and ethylene glycol)and salt, (b) one or more solvents, salt and detergent, (c) detergent,or (d) detergent and salt. Pretreatment reagents are known in the art,and such pretreatment can be employed, e.g., as used for assays onAbbott TDx, AxSYM®, and ARCHITECT® analyzers (Abbott Laboratories,Abbott Park, Ill.), as described in the literature (see, e.g., Yatscoffet al., Abbott TDx Monoclonal Antibody Assay Evaluated for MeasuringCyclosporine in Whole Blood, Clin. Chem. 36: 1969-1973 (1990), andWallemacq et al., Evaluation of the New AxSYM Cyclosporine Assay:Comparison with TDx Monoclonal Whole Blood and EMIT Cyclosporine Assays,Clin. Chem. 45: 432-435 (1999)), and/or as commercially available.Additionally, pretreatment can be done as described in Abbott's U.S.Pat. No. 5,135,875, European Pat. Pub. No. 0 471 293, U.S. ProvisionalPat. App. 60/878,017, filed Dec. 29, 2006, and U.S. Pat. App. Pub. No.2008/0020401 (incorporated by reference in its entirety for itsteachings regarding pretreatment). The pretreatment reagent can be aheterogeneous agent or a homogeneous agent.

With use of a heterogeneous pretreatment reagent, the pretreatmentreagent precipitates analyte binding protein (e.g., protein that canbind to sFlt-1 or a fragment thereof) present in the sample. Such apretreatment step comprises removing any analyte binding protein byseparating from the precipitated analyte binding protein the supernatantof the mixture formed by addition of the pretreatment agent to sample.In such an assay, the supernatant of the mixture absent any bindingprotein is used in the assay, proceeding directly to the antibodycapture step.

With use of a homogeneous pretreatment reagent there is no suchseparation step. The entire mixture of test sample and pretreatmentreagent are contacted with a labeled specific binding partner for sFlt-1(or a fragment thereof), such as a labeled anti-sFlt-1 monoclonalantibody (or an antigenically reactive fragment thereof). Thepretreatment reagent employed for such an assay typically is diluted inthe pretreated test sample mixture, either before or during capture bythe first specific binding partner. Despite such dilution, a certainamount of the pretreatment reagent (for example, 5 M methanol and/or 0.6Methylene glycol) is still present (or remains) in the test samplemixture during capture.

In a heterogeneous format, after the test sample is obtained from asubject, a first mixture is prepared. The mixture contains the testsample being assessed for sFlt-1 (or fragments thereof) and a firstspecific binding partner, wherein the first specific binding partner andany sFlt-1 contained in the test sample form a first specific bindingpartner-sFlt-1 complex. Preferably, the first specific binding partneris an anti-sFlt-1 antibody or a fragment thereof. The order in which thetest sample and the first specific binding partner are added to form themixture is not critical. Preferably, the first specific binding partneris immobilized on a solid phase. The solid phase used in the immunoassay(for the first specific binding partner and, optionally, the secondspecific binding partner) can be any solid phase known in the art, suchas, but not limited to, a magnetic particle, a bead, a test tube, amicrotiter plate, a cuvette, a membrane, a scaffolding molecule, a film,a filter paper, a disc and a chip.

After the mixture containing the first specific binding partner-sFlt-1complex is formed, any unbound sFlt-1 is removed from the complex usingany technique known in the art. For example, the unbound sFlt-1 can beremoved by washing. Desirably, however, the first specific bindingpartner is present in excess of any sFlt-1 present in the test sample,such that all sFlt-1 that is present in the test sample is bound by thefirst specific binding partner.

After any unbound sFlt-1 is removed, a second specific binding partneris added to the mixture to form a first specific bindingpartner-sFlt-1-second specific binding partner complex. The secondspecific binding partner is preferably an anti-sFlt-1 antibody thatbinds to an epitope on sFlt-1 that differs from the epitope on sFlt-1bound by the first specific binding partner. Moreover, also preferably,the second specific binding partner is labeled with or contains adetectable label as described above.

Any suitable detectable label as is known in the art can be used. Forexample, the detectable label can be a radioactive label (such as ³H,¹²⁵I, ³⁵S, ¹⁴C, ³²P, and ³³P), an enzymatic label (such as horseradishperoxidase, alkaline peroxidase, glucose 6-phosphate dehydrogenase, andthe like), a chemiluminescent label (such as acridinium esters,thioesters, or sulfonamides; luminol, isoluminol, phenanthridiniumesters, and the like), a fluorescent label (such as fluorescein (e.g.,5-fluorescein, 6-carboxyfluorescein, 3′6-carboxyfluorescein,5(6)-carboxyfluorescein, 6-hexachloro-fluorescein,6-tetrachlorofluorescein, fluorescein isothiocyanate, and the like)),rhodamine, phycobiliproteins, R-phycoerythrin, quantum dots (e.g., zincsulfide-capped cadmium selenide), a thermometric label, or animmuno-polymerase chain reaction label. An introduction to labels,labeling procedures and detection of labels is found in Polak and VanNoorden, Introduction to Immunocytochemistry, 2^(nd) ed., SpringerVerlag, N.Y. (1997), and in Haugland, Handbook of Fluorescent Probes andResearch Chemicals (1996), which is a combined handbook and cataloguepublished by Molecular Probes, Inc., Eugene, Oreg. A fluorescent labelcan be used in FPIA (see, e.g., U.S. Pat. Nos. 5,593,896, 5,573,904,5,496,925, 5,359,093, and 5,352,803, which are hereby incorporated byreference in their entireties). An acridinium compound can be used as adetectable label in a homogeneous chemiluminescent assay (see, e.g.,Adamczyk et al., Bioorg. Med. Chem. Lett. 16: 1324-1328 (2006); Adamczyket al., Bioorg. Med. Chem. Lett. 4: 2313-2317 (2004); Adamczyk et al.,Biorg. Med. Chem. Lett. 14: 3917-3921 (2004); and Adamczyk et al., Org.Lett. 5: 3779-3782 (2003)).

A preferred acridinium compound is an acridinium-9-carboxamide. Methodsfor preparing acridinium 9-carboxamides are described in Mattingly, J.Biolumin. Chemilumin. 6: 107-114 (1991); Adamczyk et al., J. Org. Chem.63: 5636-5639 (1998); Adamczyk et al., Tetrahedron 55: 10899-10914(1999); Adamczyk et al., Org. Lett. 1: 779-781 (1999); Adamczyk et al.,Bioconjugate Chem. 11: 714-724 (2000); Mattingly et al., In LuminescenceBiotechnology: Instruments and Applications; Dyke, K. V. Ed.; CRC Press:Boca Raton, pp. 77-105 (2002); Adamczyk et al., Org. Lett. 5: 3779-3782(2003); and U.S. Pat. Nos. 5,468,646, 5,543,524 and 5,783,699 (each ofwhich is incorporated herein by reference in its entirety for itsteachings regarding same).

Another preferred acridinium compound is an acridinium-9-carboxylatearyl ester. An example of an acridinium-9-carboxylate aryl ester offormula II is 10-methyl-9-(phenoxycarbonyl)acridinium fluorosulfonate(available from Cayman Chemical, Ann Arbor, Mich.). Methods forpreparing acridinium 9-carboxylate aryl esters are described in McCapraet al., Photochem. Photobiol. 4: 1111-21 (1965); Razavi et al.,Luminescence 15: 245-249 (2000); Razavi et al., Luminescence 15: 239-244(2000); and U.S. Pat. No. 5,241,070 (each of which is incorporatedherein by reference in its entirety for its teachings regarding same).Such acridinium-9-carboxylate aryl esters are efficient chemiluminescentindicators for hydrogen peroxide produced in the oxidation of an analyteby at least one oxidase in terms of the intensity of the signal and/orthe rapidity of the signal. The course of the chemiluminescent emissionfor the acridinium-9-carboxylate aryl ester is completed rapidly, i.e.,in under 1 second, while the acridinium-9-carboxamide chemiluminescentemission extends over 2 seconds. Acridinium-9-carboxylate aryl ester,however, loses its chemiluminescent properties in the presence ofprotein. Therefore, its use requires the absence of protein duringsignal generation and detection. Methods for separating or removingproteins in the sample are well-known to those skilled in the art andinclude, but are not limited to, ultrafiltration, extraction,precipitation, dialysis, chromatography, and/or digestion (see, e.g.,Wells, High Throughput Bioanalytical Sample Preparation. Methods andAutomation Strategies, Elsevier (2003)). The amount of protein removedor separated from the test sample can be about 40%, about 45%, about50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%,about 85%, about 90%, or about 95%. Further details regardingacridinium-9-carboxylate aryl ester and its use are set forth in U.S.patent application Ser. No. 11/697,835, filed Apr. 9, 2007.Acridinium-9-carboxylate aryl esters can be dissolved in any suitablesolvent, such as degassed anhydrous N,N-dimethylformamide (DMF) oraqueous sodium cholate.

Chemiluminescent assays can be performed in accordance with the methodsdescribed in Adamczyk et al., Anal. Chim. Acta 579(1): 61-67 (2006).While any suitable assay format can be used, a microplatechemiluminometer (Mithras LB-940, Berthold Technologies U.S.A., LLC, OakRidge, Tenn.) enables the assay of multiple samples of small volumesrapidly. The chemiluminometer can be equipped with multiple reagentinjectors using 96-well black polystyrene microplates (Costar #3792).Each sample can be added into a separate well, followed by thesimultaneous/sequential addition of other reagents as determined by thetype of assay employed. Desirably, the formation of pseudobases inneutral or basic solutions employing an acridinium aryl ester isavoided, such as by acidification. The chemiluminescent response is thenrecorded well-by-well. In this regard, the time for recording thechemiluminescent response will depend, in part, on the delay between theaddition of the reagents and the particular acridinium employed.

The order in which the test sample and the specific binding partner(s)are added to form the mixture for chemiluminescent assay is notcritical. If the first specific binding partner is detectably labeledwith an acridinium compound, detectably labeled first specific bindingpartner-sFlt-1 complexes form. Alternatively, if a second specificbinding partner is used and the second specific binding partner isdetectably labeled with an acridinium compound, detectably labeled firstspecific binding partner-sFlt-1-second specific binding partnercomplexes form. Any unbound specific binding partner, whether labeled orunlabeled, can be removed from the mixture using any technique known inthe art, such as washing.

Hydrogen peroxide can be generated in situ in the mixture or provided orsupplied to the mixture before, simultaneously with, or after theaddition of an above-described acridinium compound. Hydrogen peroxidecan be generated in situ in a number of ways such as would be apparentto one skilled in the art.

Alternatively, a source of hydrogen peroxide can be simply added to themixture. For example, the source of the hydrogen peroxide can be one ormore buffers or other solutions that are known to contain hydrogenperoxide. In this regard, a solution of hydrogen peroxide can simply beadded.

Upon the simultaneous or subsequent addition of at least one basicsolution to the sample, a detectable signal, namely, a chemiluminescentsignal, indicative of the presence of sFlt-1 is generated. The basicsolution contains at least one base and has a pH greater than or equalto 10, preferably, greater than or equal to 12. Examples of basicsolutions include, but are not limited to, sodium hydroxide, potassiumhydroxide, calcium hydroxide, ammonium hydroxide, magnesium hydroxide,sodium carbonate, sodium bicarbonate, calcium hydroxide, calciumcarbonate, and calcium bicarbonate. The amount of basic solution addedto the sample depends on the concentration of the basic solution. Basedon the concentration of the basic solution used, one skilled in the artcan easily determine the amount of basic solution to add to the sample.

The chemiluminescent signal that is generated can be detected usingroutine techniques known to those skilled in the art. Based on theintensity of the signal generated, the amount of sFlt-1 in the samplecan be quantified. Specifically, the amount of sFlt-1 in the sample isproportional to the intensity of the signal generated. The amount ofsFlt-1 present can be quantified by comparing the amount of lightgenerated to a standard curve for sFlt-1 or by comparison to a referencestandard. The standard curve can be generated using serial dilutions orsolutions of known concentrations of sFlt-1 by mass spectroscopy,gravimetric methods, and other techniques known in the art.

sFlt-1 immunoassays generally can be conducted using any format known inthe art, such as, but not limited to, a sandwich format, as furtherdescribed in U.S. Provisional Patent Application No. 60/981,473 (the'473 application), which was filed on Oct. 19, 2007, and which is herebyincorporated by reference. Specifically, in one format at least twoantibodies are employed to separate and quantify sFlt-1, such as humansFlt-1, or a fragment thereof in a sample. More specifically, the atleast two antibodies bind to certain different epitopes on sFlt-1 (or afragment thereof) forming an immune complex, which is referred to as a“sandwich.” Generally, in the immunoassays one or more antibodies can beused to capture the sFlt-1 (or a fragment thereof) in the test sample(these antibodies are frequently referred to as a “capture” antibody or“capture” antibodies) and one or more antibodies can be used to bind adetectable (namely, quantifiable) label to the sandwich (theseantibodies are frequently referred to as the “detection antibody,” the“detection antibodies,” the “conjugate,” or the “conjugates”).

Generally speaking, a sample being tested for (for example, suspected ofcontaining) sFlt-1 (or a fragment thereof) can be contacted with atleast one capture antibody (or antibodies) and at least one detectionantibody (which can be a second detection antibody or a third detectionantibody) either simultaneously or sequentially and in any order. Forexample, the test sample can be first contacted with at least onecapture antibody and then (sequentially) with at least one detectionantibody. Alternatively, the test sample can be first contacted with atleast one detection antibody and then (sequentially) with at least onecapture antibody. In yet another alternative, the test sample can becontacted simultaneously with a capture antibody and a detectionantibody.

In the sandwich assay format, a sample suspected of containing sFlt-1(or a fragment thereof) is first brought into contact with an at leastone first capture antibody under conditions that allow the formation ofa first antibody/sFlt-1 complex. If more than one capture antibody isused, a first multiple capture antibody/sFlt-1 complex is formed. In asandwich assay, the antibodies, preferably, the at least one captureantibody, are used in molar excess amounts of the maximum amount ofsFlt-1 (or a fragment thereof) expected in the test sample. For example,from about 5 μg to about 1 mg of antibody per mL of buffer (e.g.,microparticle coating buffer) can be used.

Competitive inhibition immunoassays, which are often used to measuresmall analytes because binding by only one antibody is required,comprise sequential and classic formats. In a sequential competitiveinhibition immunoassay a capture monoclonal antibody to an analyte ofinterest is coated onto a well of a microtiter plate. When the samplecontaining the analyte of interest is added to the well, the analyte ofinterest binds to the capture monoclonal antibody. After washing, aknown amount of labeled (e.g., biotin or horseradish peroxidase (HRP))analyte is added to the well. A substrate for an enzymatic label isnecessary to generate a signal. An example of a suitable substrate forHRP is 3,3′,5,5′-tetramethylbenzidine (TMB). After washing, the signalgenerated by the labeled analyte is measured and is inverselyproportional to the amount of analyte in the sample. In a classiccompetitive inhibition immunoassay a monoclonal antibody to an analyteof interest is coated onto a well of a microtiter plate. However, unlikethe sequential competitive inhibition immunoassay, the sample and thelabeled analyte are added to the well at the same. Any analyte in thesample competes with labeled analyte for binding to the capturemonoclonal antibody. After washing, the signal generated by the labeledanalyte is measured and is inversely proportional to the amount ofanalyte in the sample.

Optionally, prior to contacting the test sample with the at least onecapture antibody (for example, the first capture antibody), the at leastone capture antibody can be bound to a solid support, which facilitatesthe separation of the first antibody/sFlt-1 (or a fragment thereof)complex from the test sample. The substrate to which the captureantibody is bound can be any suitable solid support or solid phase thatfacilitates separation of the capture antibody-analyte complex from thesample. Examples include a well of a plate, such as a microtiter plate,a test tube, a porous gel (e.g., silica gel, agarose, dextran, orgelatin), a polymeric film (e.g., polyacrylamide), beads (e.g.,polystyrene beads or magnetic beads), a strip of a filter/membrane(e.g., nitrocellulose or nylon), microparticles (e.g., latex particles,magnetizable microparticles (e.g., microparticles having ferric oxide orchromium oxide cores and homo- or hetero-polymeric coats and radii ofabout 1-10 microns). The substrate can comprise a suitable porousmaterial with a suitable surface affinity to bind antigens andsufficient porosity to allow access by detection antibodies. Amicroporous material is generally preferred, although a gelatinousmaterial in a hydrated state can be used. Such porous substrates arepreferably in the form of sheets having a thickness of about 0.01 toabout 0.5 mm, preferably about 0.1 mm. While the pore size may varyquite a bit, preferably the pore size is from about 0.025 to about 15microns, more preferably from about 0.15 to about 15 microns. Thesurface of such substrates can be activated by chemical processes thatcause covalent linkage of an antibody to the substrate. Irreversiblebinding, generally by adsorption through hydrophobic forces, of theantigen or the antibody to the substrate results; alternatively, achemical coupling agent or other means can be used to bind covalentlythe antibody to the substrate, provided that such binding does notinterfere with the ability of the antibody to bind to sFlt-1.

Alternatively, the antibody can be bound with microparticles, which havebeen previously coated with streptavidin or biotin (e.g., usingPower-Bind™-SA-MP streptavidin-coated microparticles (Seradyn,Indianapolis, Ind.)) or anti-species-specific monoclonal antibodies. Ifnecessary, the substrate can be derivatized to allow reactivity withvarious functional groups on the antibody. Such derivatization requiresthe use of certain coupling agents, examples of which include, but arenot limited to, maleic anhydride, N-hydroxysuccinimide, and1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. If desired, one or morecapture reagents, such as antibodies (or fragments thereof), each ofwhich is specific for sFlt-1 can be attached to solid phases indifferent physical or addressable locations (e.g., such as in a biochipconfiguration (see, e.g., U.S. Pat. No. 6,225,047, Int'l Pat. App. Pub.No. WO 99/51773; U.S. Pat. No. 6,329,209; Intl Pat. App. Pub. No. WO00/56934, and U.S. Pat. No. 5,242,828). If the capture reagent isattached to a mass spectrometry probe as the solid support, the amountof sFlt-1 bound to the probe can be detected by laserdesorptionionization mass spectrometry. Alternatively, a single columncan be packed with different beads, which are derivatized with the oneor more capture reagents, thereby capturing the sFlt-1 in a single place(see, antibody derivatized, bead-based technologies, e.g., the xMAPtechnology of Luminex (Austin, Tex.)).

After the test sample being assayed for sFlt-1 (or a fragment thereof)is brought into contact with at least one capture antibody (for example,the first capture antibody), the mixture is incubated in order to allowfor the formation of a first antibody (or multiple antibody)-sFlt-1 (ora fragment thereof) complex. The incubation can be carried out at a pHof from about 4.5 to about 10.0, at a temperature of from about 2° C. toabout 45° C., and for a period from at least about one (1) minute toabout eighteen (18) hours, preferably from about 1 to about 24 minutes,most preferably for about 4 to about 18 minutes. The immunoassaydescribed herein can be conducted in one step (meaning the test sample,at least one capture antibody and at least one detection antibody areall added sequentially or simultaneously to a reaction vessel) or inmore than one step, such as two steps, three steps, etc.

After formation of the (first or multiple) capture antibody/sFlt-1 (or afragment thereof) complex, the complex is then contacted with at leastone detection antibody (under conditions which allow for the formationof a (first or multiple) capture antibody/sFlt-1 (or a fragmentthereof)/second antibody detection complex). The at least one detectionantibody can be the second, third, fourth, etc. antibodies used in theimmunoassay. If the capture antibody/sFlt-1 (or a fragment thereof)complex is contacted with more than one detection antibody, then a(first or multiple) capture antibody/sFlt-1 (or a fragmentthereof)/(multiple) detection antibody complex is formed. As with thecapture antibody (e.g., the first capture antibody), when the at leastsecond (and subsequent) detection antibody is brought into contact withthe capture antibody/sFlt-1 (or a fragment thereof) complex, a period ofincubation under conditions similar to those described above is requiredfor the formation of the (first or multiple) capture antibody/sFlt-1 (ora fragment thereof)/(second or multiple) detection antibody complex.Preferably, at least one detection antibody contains a detectable label.The detectable label can be bound to the at least one detection antibody(e.g., the second detection antibody) prior to, simultaneously with, orafter the formation of the (first or multiple) capture antibody/sFlt-1(or a fragment thereof)/(second or multiple) detection antibody complex.Any detectable label known in the art can be used (see discussion above,including Polak and Van Noorden (1997) and Haugland (1996)).

The detectable label can be bound to the antibodies either directly orthrough a coupling agent. An example of a coupling agent that can beused is EDAC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide,hydrochloride), which is commercially available from Sigma-Aldrich, St.Louis, Mo. Other coupling agents that can be used are known in the art.Methods for binding a detectable label to an antibody are known in theart. Additionally, many detectable labels can be purchased orsynthesized that already contain end groups that facilitate the couplingof the detectable label to the antibody, such as CPSP-Acridinium Ester(i.e., 9-[N-tosyl-N-(3-carboxypropyl)]-10-(3-sulfopropyl)acridiniumcarboxamide) or SPSP-Acridinium Ester (i.e.,N10-(3-sulfopropyl)-N-(3-sulfopropyl)-acridinium-9-carboxamide).

The (first or multiple) capture antibody/sFlt-1/(second or multiple)detection antibody complex can be, but does not have to be, separatedfrom the remainder of the test sample prior to quantification of thelabel. For example, if the at least one capture antibody (e.g., thefirst capture antibody) is bound to a solid support, such as a well or abead, separation can be accomplished by removing the fluid (of the testsample) from contact with the solid support. Alternatively, if the atleast first capture antibody is bound to a solid support, it can besimultaneously contacted with the sFlt-1-containing sample and the atleast one second detection antibody to form a first (multiple)antibody/sFlt-1/second (multiple) antibody complex, followed by removalof the fluid (test sample) from contact with the solid support. If theat least one first capture antibody is not bound to a solid support,then the (first or multiple) capture antibody/sFlt-1/(second ormultiple) detection antibody complex does not have to be removed fromthe test sample for quantification of the amount of the label.

After formation of the labeled capture antibody/sFlt-1/detectionantibody complex (e.g., the first capture antibody/sFlt-1/seconddetection antibody complex), the amount of label in the complex isquantified using techniques known in the art. For example, if anenzymatic label is used, the labeled complex is reacted with a substratefor the label that gives a quantifiable reaction such as the developmentof color. If the label is a radioactive label, the label is quantifiedusing a scintillation counter. If the label is a fluorescent label, thelabel is quantified by stimulating the label with a light of one color(which is known as the “excitation wavelength”) and detecting anothercolor (which is known as the “emission wavelength”) that is emitted bythe label in response to the stimulation. If the label is achemiluminescent label, the label is quantified by detecting the lightemitted either visually or by using luminometers, x-ray film, high speedphotographic film, a CCD camera, etc. Once the amount of the label inthe complex has been quantified, the concentration of sFlt-1 or afragment thereof in the test sample is determined by use of a standardcurve that has been generated using serial dilutions of sFlt-1 or afragment thereof of known concentration. Other than using serialdilutions of sFlt-1 or a fragment thereof, the standard curve can begenerated gravimetrically, by mass spectroscopy and by other techniquesknown in the art.

In a chemiluminescent microparticle assay employing the ARCHITECT®analyzer, the conjugate diluent pH should be about 6.0+/−0.2, themicroparticle coating buffer should be maintained at room temperature(i.e., at about 17 to about 27° C.), the microparticle coating buffer pHshould be about 6.5+/−0.2, and the microparticle diluent pH should beabout 7.8+/−0.2. Solids preferably are less than about 0.2%, such asless than about 0.15%, less than about 0.14%, less than about 0.13%,less than about 0.12%, or less than about 0.11%, such as about 0.10%.

FPIAs are based on competitive binding immunoassay principles. Afluorescently labeled compound, when excited by a linearly polarizedlight, will emit fluorescence having a degree of polarization inverselyproportional to its rate of rotation. When a fluorescently labeledtracer-antibody complex is excited by a linearly polarized light, theemitted light remains highly polarized because the fluorophore isconstrained from rotating between the time light is absorbed and thetime light is emitted. When a “free” tracer compound (i.e., a compoundthat is not bound to an antibody) is excited by linearly polarizedlight, its rotation is much faster than the correspondingtracer-antibody conjugate produced in a competitive binding immunoassay.FPIAs are advantageous over RIAs inasmuch as there are no radioactivesubstances requiring special handling and disposal. In addition, FPIAsare homogeneous assays that can be easily and rapidly performed.

A commercially available anti-sFlt-1 antibody can be used in the methodsof assay and kits there of. Commercially available anti-sFlt-1antibodies include those available from Santa Cruz Biotechnology, Inc.,and R&D Systems and those used in the Examples herein. Preferably, suchcommercially available antibodies are used as detection antibodies.

Any suitable control composition can be used in the sFlt-1 immunoassays.The control composition generally comprises sFlt-1 and any desirableadditives.

Thus, in view of the above, a method of determining the presence, amountor concentration of sFlt-1 or a fragment thereof in a test sample isprovided. The method comprises assaying the test sample for sFlt-1 (or afragment thereof) by an immunoassay employing at least one antibody andat least one detectable label and comprising comparing a signalgenerated by the detectable label as a direct or indirect indication ofthe presence, amount or concentration of sFlt-1 in the test sample to asignal generated as a direct or indirect indication of the presence,amount or concentration of sFlt-1 in a calibrator. The calibrator isoptionally part of a series of calibrators in which each of thecalibrators differs from the other calibrators in the series by theconcentration of sFlt-1. One of the at least one antibody is an isolatedantibody, which specifically binds to sFlt-1 or a fragment thereof, andwhich has (i) a variable heavy domain region comprising the amino acidsequence of SEQ ID NO: 2, (ii) a variable light domain region comprisingthe amino acid sequence of SEQ ID NO: 4, or (iii) a variable heavydomain region comprising the amino acid sequence of SEQ ID NO: 2 and avariable light domain region comprising the amino acid sequence of SEQID NO: 4.

The method can comprise (i) contacting the test sample with at least onecapture antibody, which binds to an epitope on sFlt-1 (or a fragmentthereof), so as to form a capture antibody/sFlt-1 (or a fragmentthereof) complex, (ii) contacting the capture antibody/sFlt-1 (or afragment thereof) complex with at least one detection antibody, whichcomprises a detectable label and binds to an epitope on sFlt-1 (or afragment thereof) that is not bound by the capture antibody, to form acapture antibody/sFlt-1 (or a fragment thereof)/detection antibodycomplex, and (iii) determining the amount of sFlt-1 (or a fragmentthereof) in the test sample based on the signal generated by thedetectable label in the capture antibody/sFlt-1 (or a fragmentthereof)/detection antibody complex formed in (ii).

Alternatively, the method can comprise (i) contacting the test samplewith at least one capture antibody, which binds to an epitope on sFlt-1(or a fragment thereof) so as to form a capture antibody/sFlt-1 (or afragment thereof) complex, and simultaneously or sequentially, in eitherorder, contacting the test sample with detectably labeled sFlt-1 (or afragment thereof), which can compete with any sFlt-1 (or a fragmentthereof) in the test sample for binding to the at least one captureantibody. Any sFlt-1 (or a fragment thereof) present in the test sampleand the detectably labeled sFlt-1 compete with each other to form acapture antibody/sFlt-1 (or a fragment thereof) complex and a captureantibody/detectably labeled sFlt-1 (or a fragment thereof) complex,respectively. The method further comprises (ii) determining thepresence, amount or concentration of sFlt-1 in the test sample based onthe signal generated by the detectable label in the captureantibody/detectably labeled sFlt-1 (or a fragment thereof) complexformed in (ii). The signal generated by the detectable label in thecapture antibody/detectably labeled sFlt-1 (or a fragment thereof)complex is inversely proportional to the amount or concentration ofsFlt-1 in the test sample.

The above methods can further comprise simultaneously or sequentially,in either order, determining the amount or concentration of VEGF (or afragment thereof) and/or P1GF (or a fragment thereof) in the testsample. The method comprises assaying the test sample for VEGF (or afragment thereof) and/or P1GF (or a fragment thereof) by an assayemploying at least one specific binding partner for VEGF (or a fragmentthereof) and/or at least one specific binding partner for P1GF (or afragment thereof), respectively, and at least one detectable label andcomprising comparing a signal generated by the detectable label as adirect or indirect indication of the amount or concentration of VEGF (ora fragment thereof) and/or P1GF (or a fragment thereof) in the testsample to a signal generated as a direct or indirect indication of theamount or concentration of VEGF (or a fragment thereof) and/or P1GF (ora fragment thereof), respectively, in a control or calibrator. Thecalibrator is optionally part of a series of calibrators in which eachof the calibrators differs from the other calibrators in the series bythe concentration of VEGF or P1GF, respectively.

The method can further comprise diagnosing, prognosticating, orassessing the efficacy of a therapeutic/prophylactic treatment of apatient from whom the test sample was obtained. If the method furthercomprises assessing the efficacy of a therapeutic/prophylactic treatmentof the patient from whom the test sample was obtained, the methodoptionally further comprises modifying the therapeutic/prophylactictreatment of the patient as needed to improve efficacy. The method canbe adapted for use in an automated system or a semi-automated system.

Generally, a predetermined level can be employed as a benchmark againstwhich to assess results obtained upon assaying a test sample for sFlt-1or a fragment thereof. Generally, in making such a comparison, thepredetermined level is obtained by running a particular assay asufficient number of times and under appropriate conditions such that alinkage or association of analyte presence, amount or concentration witha particular stage or endpoint of a disease, disorder or condition(e.g., preeclampsia or cardiovascular disease) or with particularindicia can be made. Typically, the predetermined level is obtained withassays of reference subjects (or populations of subjects). A level ofsFlt-1 greater than 2 ng/mL, for example, can be indicative ofpreeclampsia or eclampsia. The sFlt-1 measured can include fragmentsthereof, degradation products thereof, and/or enzymatic cleavageproducts thereof.

In particular, with respect to a predetermined level as employed formonitoring disease progression and/or treatment, the amount orconcentration of sFlt-1 or a fragment thereof may be “unchanged,”“favorable” (or “favorably altered”), or “unfavorable” (or “unfavorablyaltered”). “Elevated” or “increased” refers to an amount or aconcentration in a test sample that is higher than a typical or normallevel or range (e.g., predetermined level), or is higher than anotherreference level or range (e.g., earlier or baseline sample). The term“lowered” or “reduced” refers to an amount or a concentration in a testsample that is lower than a typical or normal level or range (e.g.,predetermined level), or is lower than another reference level or range(e.g., earlier or baseline sample). The term “altered” refers to anamount or a concentration in a sample that is altered (increased ordecreased) over a typical or normal level or range (e.g., predeterminedlevel), or over another reference level or range (e.g., earlier orbaseline sample).

The typical or normal level or range for sFlt-1 is defined in accordancewith standard practice. Because the levels of sFlt-1 in some instanceswill be very low, a so-called altered level or alteration can beconsidered to have occurred when there is any net change as compared tothe typical or normal level or range, or reference level or range, thatcannot be explained by experimental error or sample variation. Thus, thelevel measured in a particular sample will be compared with the level orrange of levels determined in similar samples from a so-called normalsubject. In this context, a “normal subject” is an individual with nodetectable preeclampsia or cardiovascular disease, for example, and a“normal” (sometimes termed “control”) patient or population is/areone(s) that exhibit(s) no detectable preeclampsia or cardiovasculardisease, respectively, for example. Furthermore, given that sFlt-1 isnot routinely found at a high level in the majority of the humanpopulation, a “normal subject” can be considered an individual with nosubstantial detectable increased or elevated amount or concentration ofsFlt-1, and a “normal” (sometimes termed “control”) patient orpopulation is/are one(s) that exhibit(s) no substantial detectableincreased or elevated amount or concentration of sFlt-1. An “apparentlynormal subject” is one in which sFlt-1 has not been or is beingassessed. The level of an analyte is said to be “elevated” when theanalyte is normally undetectable (e.g., the normal level is zero, orwithin a range of from about 25 to about 75 percentiles of normalpopulations), but is detected in a test sample, as well as when theanalyte is present in the test sample at a higher than normal level.Thus, inter alia, the disclosure provides a method of screening for asubject having, or at risk of having, preeclampsia or a cardiovasculardisease, or cancer, for example, as defined herein.

The method of assay can also involve the assay of other markers and thelike. For example, the method of assay can also involve the assay ofVEGF (decrease in level of) and/or P1GF (decrease in level of).Alternatively or additionally, the method of assay can involve themeasurement of an obesity factor (e.g., body mass index (BMI); see,e.g., Sogin et al., U.S. Pat. App. Pub. No. 2008/0071151, which waspublished on Mar. 20, 2008, and is hereby incorporated by reference withregard to its teachings regarding same), and/or gestational age (GA).Accordingly, the methods described herein also can be used to determinewhether or not a subject has or is at risk of developing preeclampsia,or a cardiovascular disease, or cancer. Specifically, such a method cancomprise the steps of:

(a) determining the concentration or amount in a test sample from asubject of sFlt-1 (or a fragment thereof) (e.g., using the methodsdescribed herein, or methods known in the art); and

(b) comparing the concentration or amount of sFlt-1 (or a fragmentthereof) determined in step (a) with a predetermined level, wherein, ifthe concentration or amount of sFlt-1 determined in step (a) isfavorable with respect to a predetermined level, then the subject isdetermined not to have or be at risk for preeclampsia or acardiovascular disease. However, if the concentration or amount ofsFlt-1 determined in step (a) is unfavorable with respect to thepredetermined level, then the subject is determined to have or be atrisk for preeclampsia or a cardiovascular disease or cancer.

Additionally, provided herein is method of monitoring the progression ofdisease in a subject. Optimally the method comprising the steps of:

(a) determining the concentration or amount in a test sample from asubject of sFlt-1;

(b) determining the concentration or amount in a later test sample fromthe subject of sFlt-1; and

(c) comparing the concentration or amount of sFlt-1 as determined instep (b) with the concentration or amount of sFlt-1 determined in step(a), wherein if the concentration or amount determined in step (b) isunchanged or is unfavorable when compared to the concentration or amountof sFlt-1 determined in step (a), then the disease in the subject isdetermined to have continued, progressed or worsened. By comparison, ifthe concentration or amount of sFlt-1 as determined in step (b) isfavorable when compared to the concentration or amount of sFlt-1 asdetermined in step (a), then the disease in the subject is determined tohave discontinued, regressed or improved.

Optionally, the method further comprises comparing the concentration oramount of sFlt-1 as determined in step (b), for example, with apredetermined level. Further, optionally the method comprises treatingthe subject with one or more pharmaceutical compositions for a period oftime if the comparison shows that the concentration or amount of sFlt-1as determined in step (b), for example, is unfavorably altered withrespect to the predetermined level.

Still further, the methods can be used to monitor treatment in a subjectreceiving treatment with one or more pharmaceutical compositions.Specifically, such methods involve providing a first test sample from asubject before the subject has been administered one or morepharmaceutical compositions. Next, the concentration or amount in afirst test sample from a subject of sFlt-1 is determined (e.g., usingthe methods described herein or as known in the art). After theconcentration or amount of sFlt-1 is determined, optionally theconcentration or amount of sFlt-1 is then compared with a predeterminedlevel. If the concentration or amount of sFlt-1 as determined in thefirst test sample is lower than the predetermined level, then thesubject is not treated with one or more pharmaceutical compositions.However, if the concentration or amount of sFlt-1 as determined in thefirst test sample is higher than the predetermined level, then thesubject is treated with one or more pharmaceutical compositions for aperiod of time. The period of time that the subject is treated with theone or more pharmaceutical compositions can be determined by one skilledin the art (for example, the period of time can be from about seven (7)days to about two years, preferably from about fourteen (14) days toabout one (1) year).

During the course of treatment with the one or more pharmaceuticalcompositions, second and subsequent test samples are then obtained fromthe subject. The number of test samples and the time in which said testsamples are obtained from the subject are not critical. For example, asecond test sample could be obtained seven (7) days after the subject isfirst administered the one or more pharmaceutical compositions, a thirdtest sample could be obtained two (2) weeks after the subject is firstadministered the one or more pharmaceutical compositions, a fourth testsample could be obtained three (3) weeks after the subject is firstadministered the one or more pharmaceutical compositions, a fifth testsample could be obtained four (4) weeks after the subject is firstadministered the one or more pharmaceutical compositions, etc.

After each second or subsequent test sample is obtained from thesubject, the concentration or amount of sFlt-1 is determined in thesecond or subsequent test sample is determined (e.g., using the methodsdescribed herein or as known in the art). The concentration or amount ofsFlt-1 as determined in each of the second and subsequent test samplesis then compared with the concentration or amount of sFlt-1 asdetermined in the first test sample (e.g., the test sample that wasoriginally optionally compared to the predetermined level). If theconcentration or amount of sFlt-1 as determined in step (c) is favorablewhen compared to the concentration or amount of sFlt-1 as determined instep (a), then the disease in the subject is determined to havediscontinued, regressed or improved, and the subject should continue tobe administered the one or pharmaceutical compositions of step (b).However, if the concentration or amount determined in step (c) isunchanged or is unfavorable when compared to the concentration or amountof sFlt-1 as determined in step (a), then the disease in the subject isdetermined to have continued, progressed or worsened, and the subjectshould be treated with a higher concentration of the one or morepharmaceutical compositions administered to the subject in step (b) orthe subject should be treated with one or more pharmaceuticalcompositions that are different from the one or more pharmaceuticalcompositions administered to the subject in step (b). Specifically, thesubject can be treated with one or more pharmaceutical compositions thatare different from the one or more pharmaceutical compositions that thesubject had previously received to decrease or lower said subject'ssFlt-1 level.

Generally, for assays in which repeat testing may be done (e.g.,monitoring disease progression and/or response to treatment), a secondor subsequent test sample is obtained at a period in time after thefirst test sample has been obtained from the subject. Specifically, asecond test sample from the subject can be obtained minutes, hours,days, weeks or years after the first test sample has been obtained fromthe subject. For example, the second test sample can be obtained fromthe subject at a time period of about 1 minute, about 5 minutes, about10 minutes, about 15 minutes, about 30 minutes, about 45 minutes, about60 minutes, about 2 hours, about 3 hours, about 4 hours, about 5 hours,about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours,about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours,about 24 hours, about 2 days, about 3 days, about 4 days, about 5 days,about 6 days, about 7 days, about 2 weeks, about 3 weeks, about 4 weeks,about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9weeks, about 10 weeks, about 11 weeks, about 12 weeks, about 13 weeks,about 14 weeks, about 15 weeks, about 16 weeks, about 17 weeks, about 18weeks, about 19 weeks, about 20 weeks, about 21 weeks, about 22 weeks,about 23 weeks, about 24 weeks, about 25 weeks, about 26 weeks, about 27weeks, about 28 weeks, about 29 weeks, about 30 weeks, about 31 weeks,about 32 weeks, about 33 weeks, about 34 weeks, about 35 weeks, about 36weeks, about 37 weeks, about 38 weeks, about 39 weeks, about 40 weeks,about 41 weeks, about 42 weeks, about 43 weeks, about 44 weeks, about 45weeks, about 46 weeks, about 47 weeks, about 48 weeks, about 49 weeks,about 50 weeks, about 51 weeks, about 52 weeks, about 1.5 years, about 2years, about 2.5 years, about 3.0 years, about 3.5 years, about 4.0years, about 4.5 years, about 5.0 years, about 5.5 years, about 6.0years, about 6.5 years, about 7.0 years, about 7.5 years, about 8.0years, about 8.5 years, about 9.0 years, about 9.5 years or about 10.0years after the first test sample from the subject is obtained. Whenused to monitor disease progression, the above assay can be used tomonitor the progression of disease in subjects suffering from acuteconditions. Acute conditions, also known as critical care conditions,refer to acute, life-threatening diseases or other critical medicalconditions involving, for example, the cardiovascular system orexcretory system. Typically, critical care conditions refer to thoseconditions requiring acute medical intervention in a hospital-basedsetting (including, but not limited to, the emergency room, intensivecare unit, trauma center, or other emergent care setting) oradministration by a paramedic or other field-based medical personnel.For critical care conditions, repeat monitoring is generally done withina shorter time frame, namely, minutes, hours or days (e.g., about 1minute, about 5 minutes, about 10 minutes, about 15 minutes, about 30minutes, about 45 minutes, about 60 minutes, about 2 hours, about 3hours, about 4 hours, 4 about 5 hours, about 6 hours, about 7 hours,about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours,about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21hours, about 22 hours, about 23 hours, about 24 hours, about 2 days,about 3 days, about 4 days, about 5 days, about 6 days or about 7 days),and the initial assay likewise is generally done within a shortertimeframe, e.g., about minutes, hours or days of the onset of thedisease or condition.

The assays also can be used to monitor the progression of disease insubjects suffering from chronic or non-acute conditions. Non-criticalcare or, non-acute conditions, refers to conditions other than acute,life-threatening disease or other critical medical conditions involving,for example, the cardiovascular system and/or excretory system.Typically, non-acute conditions include those of longer-term or chronicduration. For non-acute conditions, repeat monitoring generally is donewith a longer timeframe, e.g., hours, days, weeks, months or years(e.g., about 1 hour, about 2 hours, about 3 hours, about 4 hours, about5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours,about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours,about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23hours, about 24 hours, about 2 days, about 3 days, about 4 days, about 5days, about 6 days, about 7 days, about 2 weeks, about 3 weeks, about 4weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about9 weeks, about 10 weeks, about 11 weeks, about 12 weeks, about 13 weeks,about 14 weeks, about 15 weeks, about 16 weeks, about 17 weeks, about 18weeks, about 19 weeks, about 20 weeks, about 21 weeks, about 22 weeks,about 23 weeks, about 24 weeks, about 25 weeks, about 26 weeks, about 27weeks, about 28 weeks, about 29 weeks, about 30 weeks, about 31 weeks,about 32 weeks, about 33 weeks, about 34 weeks, about 35 weeks, about 36weeks, about 37 weeks, about 38 weeks, about 39 weeks, about 40 weeks,about 41 weeks, about 42 weeks, about 43 weeks, about 44 weeks, about 45weeks, about 46 weeks, about 47 weeks, about 48 weeks, about 49 weeks,about 50 weeks, about 51 weeks, about 52 weeks, about 1.5 years, about 2years, about 2.5 years, about 3.0 years, about 3.5 years, about 4.0years, about 4.5 years, about 5.0 years, about 5.5 years, about 6.0years, about 6.5 years, about 7.0 years, about 7.5 years, about 8.0years, about 8.5 years, about 9.0 years, about 9.5 years or about 10.0years), and the initial assay likewise generally is done within a longertime frame, e.g., about hours, days, months or years of the onset of thedisease or condition.

Furthermore, the above assays can be performed using a first test sampleobtained from a subject where the first test sample is obtained from onesource, such as urine, serum or plasma. Optionally the above assays canthen be repeated using a second test sample obtained from the subjectwhere the second test sample is obtained from another source. Forexample, if the first test sample was obtained from urine, the secondtest sample can be obtained from serum or plasma. The results obtainedfrom the assays using the first test sample and the second test samplecan be compared. The comparison can be used to assess the status of adisease or condition in the subject.

Moreover, the present disclosure also relates to methods of determiningwhether a subject predisposed to or suffering from a disease (e.g.,preeclampsia or a cardiovascular disease or cancer) will benefit fromtreatment. In particular, the disclosure relates to sFlt-1 companiondiagnostic methods and products. Thus, the method of “monitoring thetreatment of disease in a subject” as described herein further optimallyalso can encompass selecting or identifying candidates for therapy.

Thus, in particular embodiments, the disclosure also provides a methodof determining whether a subject having, or at risk for, preeclampsia ora cardiovascular disease or cancer is a candidate for therapy.Generally, the subject is one who has experienced some symptom ofpreeclampsia or a cardiovascular disease or cancer or who has actuallybeen diagnosed as having, or being at risk for, preeclampsia or acardiovascular disease, or cancer and/or who demonstrates an unfavorableconcentration or amount of sFlt-1 or a fragment thereof, as describedherein.

The method optionally comprises an assay as described herein, whereanalyte is assessed before and following treatment of a subject with oneor more pharmaceutical compositions (e.g., particularly with apharmaceutical related to a mechanism of action involving sFlt-1), withimmunosuppressive therapy, or by immunoabsorption therapy, withanti-angiogenic therapy, or where analyte is assessed following suchtreatment and the concentration or the amount of analyte is comparedagainst a predetermined level. An unfavorable concentration of amount ofanalyte observed following treatment confirms that the subject will notbenefit from receiving further or continued treatment, whereas afavorable concentration or amount of analyte observed followingtreatment confirms that the subject will benefit from receiving furtheror continued treatment. This confirmation assists with management ofclinical studies, and provision of improved patient care.

For instance, the assays and kits optionally can be employed wherein thetest sample is from a patient with signs and/or symptoms of an acutecoronary syndrome and the method further comprises diagnosing orprognosticating the risk of experiencing a major adverse cardiac event(MACE). Assay of sFlt-1 as described herein optionally can be employedwith other markers, for instance, in combination with cardiac troponin(I or T). Thus, further provided herein is an assay for sFlt-1 whereinthe test sample is from a patient with signs and/or symptoms of an acutecoronary syndrome, and the method further comprises diagnosing orprognosticating the risk of experiencing a major adverse cardiac event(MACE) in combination with cardiac troponin (I or T).

It goes without saying that, while certain embodiments herein areadvantageous when employed to assess preeclampsia or a cardiovasculardisease or cancer, the assays and kits also optionally can be employedto assess sFlt-1 in other diseases, disorders and conditions. Forexample, the integral role of growth factors in angiogenesis has beenwell documented. Additionally, sFlt-1 has been employed inantiangiogenic therapy in combination with immunotherapy withtumor-associated antigen (see, e.g., Cuadros et al., Cancer Research,63:5895-5901 (2003)). For this and other reasons, assays as proposedherein can be employed, among other things, in assessment of cancer andcancer treatment.

The method of assay also can be used to identify a compound thatameliorates preeclampsia, cardiovascular disease, cancer and the like.For example, a cell that expresses sFlt-1 can be contacted with acandidate compound. The level of expression of sFlt-1 in the cellcontacted with the compound can be compared to that in a control cellusing the method of assay described herein.

The methods of assay as described herein further can be carried outwhere sFlt-1 is measured along with one or more other analytes, e.g.,P/GF.

Adaptation of Kit and Method

The kit (or components thereof), as well as the method of determiningthe concentration of sFlt-1 in a test sample by an immunoassay asdescribed herein, can be adapted for use in a variety of automated andsemi-automated systems (including those wherein the solid phasecomprises a microparticle), as described, e.g., in U.S. Pat. Nos.5,089,424 and 5,006,309, and as commercially marketed, e.g., by AbbottLaboratories (Abbott Park, Ill.) as ARCHITECT®.

Some of the differences between an automated or semi-automated system ascompared to a non-automated system (e.g., ELISA) include the substrateto which the first specific binding partner (e.g., analyte antibody orcapture antibody) is attached (which can impact sandwich formation andanalyte reactivity), and the length and timing of the capture, detectionand/or any optional wash steps. Whereas a non-automated format such asan ELISA may require a relatively longer incubation time with sample andcapture reagent (e.g., about 2 hours), an automated or semi-automatedformat (e.g., ARCHITECT®, Abbott Laboratories) may have a relativelyshorter incubation time (e.g., approximately 18 minutes for ARCHITECT®).Similarly, whereas a non-automated format such as an ELISA may incubatea detection antibody such as the conjugate reagent for a relativelylonger incubation time (e.g., about 2 hours), an automated orsemi-automated format (e.g., ARCHITECT®) may have a relatively shorterincubation time (e.g., approximately 4 minutes for the ARCHITECT®).

Other platforms available from Abbott Laboratories include, but are notlimited to, AxSYM®, IMx® (see, e.g., U.S. Pat. No. 5,294,404, which ishereby incorporated by reference in its entirety), PRISM®, EIA (bead),and Quantum™ II, as well as other platforms. Additionally, the assays,kits and kit components can be employed in other formats, for example,on electrochemical or other hand-held or point-of-care assay systems.The present disclosure is, for example, applicable to the commercialAbbott Point of Care (i-STAT®, Abbott Laboratories) electrochemicalimmunoassay system that performs sandwich immunoassays. Immunosensorsand their methods of manufacture and operation in single-use testdevices are described, for example in, U.S. Pat. No. 5,063,081, U.S.Pat. App. Pub. No. 2003/0170881, U.S. Pat. App. Pub. No. 2004/0018577,U.S. Pat. App. Pub. No. 2005/0054078, and U.S. Pat. App. Pub. No.2006/0160164, which are incorporated in their entireties by referencefor their teachings regarding same.

In particular, with regard to the adaptation of an assay to the I-STAT®system, the following configuration is preferred. A microfabricatedsilicon chip is manufactured with a pair of gold amperometric workingelectrodes and a silver-silver chloride reference electrode. On one ofthe working electrodes, polystyrene beads (0.2 mm diameter) withimmobilized capture antibody are adhered to a polymer coating ofpatterned polyvinyl alcohol over the electrode. This chip is assembledinto an I-STAT® cartridge with a fluidics format suitable forimmunoassay. On a portion of the wall of the sample-holding chamber ofthe cartridge there is a layer comprising the detection antibody labeledwith alkaline phosphatase (or other label). Within the fluid pouch ofthe cartridge is an aqueous reagent that includes p-aminophenolphosphate.

In operation, a sample suspected of containing sFlt-1 is added to theholding chamber of the test cartridge and the cartridge is inserted intothe I-STAT® reader. After the second antibody (detection antibody) hasdissolved into the sample, a pump element within the cartridge forcesthe sample into a conduit containing the chip. Here it is oscillated topromote formation of the sandwich between the first capture antibody,sFlt-1, and the labeled second detection antibody. In the penultimatestep of the assay, fluid is forced out of the pouch and into the conduitto wash the sample off the chip and into a waste chamber. In the finalstep of the assay, the alkaline phosphatase label reacts withp-aminophenol phosphate to cleave the phosphate group and permit theliberated p-aminophenol to be electrochemically oxidized at the workingelectrode. Based on the measured current, the reader is able tocalculate the amount of analyte IL-18 in the sample by means of anembedded algorithm and factory-determined calibration curve.

It further goes without saying that the methods and kits as describedherein necessarily encompass other reagents and methods for carrying outthe immunoassay. For instance, encompassed are various buffers such asare known in the art and/or which can be readily prepared or optimizedto be employed, e.g., for washing, as a conjugate diluent, and/or as acalibrator diluent. An exemplary conjugate diluent is ARCHITECT®conjugate diluent employed in certain kits (Abbott Laboratories, AbbottPark, Ill.) and containing 2-(N-morpholino)ethanesulfonic acid (MES), asalt, a protein blocker, an antimicrobial agent, and a detergent. Anexemplary calibrator diluent is ARCHITECT® human calibrator diluentemployed in certain kits (Abbott Laboratories, Abbott Park, Ill.), whichcomprises a buffer containing MES, other salt, a protein blocker, and anantimicrobial agent. Additionally, as described in U.S. PatentApplication No. 61/142,048 filed Dec. 31, 2008, improved signalgeneration may be obtained, e.g., in an I-STAT® cartridge format, usinga nucleic acid sequence linked to the signal antibody as a signalamplifier.

Furthermore, the methods and kits optionally are adapted for use on anautomated or semi-automated system. Some of the differences between anautomated or semi-automated system as compared to a non-automated system(e.g., ELISA) include the substrate to which the first specific bindingpartner (e.g., analyte antigen or capture antibody) is attached (whichcan impact sandwich formation and analyte reactivity), and the lengthand timing of the capture, detection and/or any optional wash steps.Whereas a non-automated format such as an ELISA may include a relativelylonger incubation time with sample and capture reagent (e.g., about 2hours) an automated or semi-automated format (e.g., ARCHITECT®) may havea relatively shorter incubation time (e.g., approximately 18 minutes forARCHITECT®). Similarly, whereas a non-automated format such as an ELISAmay incubate a detection antibody such as the conjugate reagent for arelatively longer incubation time (e.g., about 2 hours), an automated orsemi-automated format (e.g., ARCHITECT®) may have a relatively shorterincubation time (e.g., approximately 4 minutes for the ARCHITECT®).

Anti-sFlt-1 Antibody Pharmaceutical Composition

A pharmaceutical composition comprising a therapeutically orprophylactically effective amount of an isolated antibody thatspecifically binds to sFlt-1 or a fragment thereof is also provided. Theantibody has a variable heavy domain region comprising the amino acidsequence of SEQ ID NO: 2, a variable light domain region comprising theamino acid sequence of SEQ ID NO: 4, or a variable heavy domain regioncomprising the amino acid sequence of SEQ ID NO: 2 and a variable lightdomain region comprising the amino acid sequence of SEQ ID NO: 4, and apharmaceutically acceptable carrier, diluent, and/or excipient. Suitablecarriers, diluents, and/or excipients are well-known in the art (see,e.g., Remington's Pharmaceutical Sciences, 20^(th) edition, Gennaro,editor, Lippincott, Williams & Wilkins, Philadelphia, Pa., 2000).Optionally, the composition further comprises another active agentand/or an adjuvant. The pharmaceutical composition is optionally part ofa kit comprising one or more containers in which the antibody, anotheractive agent and/or the adjuvant can be present in the same or differentcontainers.

Recombinant forms of antibodies, such as chimeric and humanizedantibodies, can be used in pharmaceutical compositions to minimize theresponse by a human patient to the antibody. When antibodies produced innon-human subjects or derived from expression of non-human antibodygenes are used therapeutically in humans, they are recognized to varyingdegrees as foreign, and an immune response may be generated in thepatient. One approach to minimize or eliminate this immune reaction isto produce chimeric antibody derivatives, namely, antibody moleculesthat combine a non-human animal variable region and a human constantregion. Such antibodies retain the epitope binding specificity of theoriginal monoclonal antibody but may be less immunogenic whenadministered to humans and, therefore, more likely to be tolerated bythe patient.

Chimeric monoclonal antibodies can be produced by recombinant DNAtechniques known in the art. For example, a gene encoding the constantregion of a non-human antibody molecule is substituted with a geneencoding a human constant region (see, for example, Int'l Pat. App. Pub.No. PCT/US86/02269, European Pat. App. 184,187, or European Pat. App.171,496).

A chimeric antibody can be further “humanized” by replacing portions ofthe variable region not involved in antigen binding with equivalentportions from human variable regions. General reviews of “humanized”chimeric antibodies can be found in Morrison, Science 229: 1202-1207(1985), and Oi et al., BioTechniques 4: 214 (1986). Such methods includeisolating, manipulating, and expressing the nucleic acid sequences thatencode all or part of an immunoglobulin variable region from at leastone of a heavy or light chain. The cDNA encoding the humanized chimericantibody, or a fragment thereof, can then be cloned into an appropriateexpression vector. Suitable “humanized” antibodies can be alternativelyproduced by complementarity determining region (CDR) substitution (see,for example, U.S. Pat. No. 5,225,539; Jones et al., Nature 321: 552-525(1986); Verhoeyan et al., Science 239: 1534 (1988); and Beidler et al.,J. Immunol. 141: 4053-4060 (1988)).

Epitope imprinting also can be used to produce a “human” antibodypolypeptide dimer that retains the binding specificity of the antibodies(e.g., hamster antibodies) specific for the human sFlt-1 orantigenically reactive fragment thereof. Briefly, a gene encoding anon-human variable region (VH) with specific binding to an antigen and ahuman constant region (CH1), is expressed in E. coli and infected with aphage library of human Vλ.Cλ genes. Phage displaying antibody fragmentsare then screened for binding to the human sFlt-1 protein. Selectedhuman Vλ genes are recloned for expression of Vλ.Cλ. chains and E. coliharboring these chains are infected with a phage library of human VHCH1genes and the library is subject to rounds of screening withantigen-coated tubes (see, e.g., Int'l Pat. App. Pub. No. WO 93/06213).

For administration to an animal, the pharmaceutical composition can beformulated for administration by a variety of routes. For example, thecomposition can be formulated for oral, topical, rectal or parenteraladministration or for administration by inhalation or spray. The term“parenteral” as used herein includes subcutaneous, intravenous,intramuscular, intrathecal, and intrasternal injection and infusiontechniques. Various diagnostic compositions and pharmaceuticalcompositions suitable for different routes of administration and methodsof preparing pharmaceutical compositions are known in the art and aredescribed, for example, in “Remington: The Science and Practice ofPharmacy” (formerly “Remington's Pharmaceutical Sciences”); Gennaro, A.,Lippincott, Williams & Wilkins, Philadelphia, Pa. (2000). Thepharmaceutical composition can be used in the treatment of variousconditions in animals, including humans.

The pharmaceutical composition preferably comprises a therapeutically orprophylactically effective amount of an anti-sFlt-1 antibody. The term“therapeutically or prophylactically effective amount” as used hereinrefers to an amount of an anti-sFlt-1 antibody needed to treat,ameliorate, inhibit the onset, delay or slow the progression, or preventa targeted disease, condition, or disorder or to exhibit a detectabletherapeutic or preventative effect. For anti-sFlt-1 antibody, thetherapeutically or prophylactically effective amount can be estimatedinitially, for example, either in cell culture assays or in animalmodels, usually in rodents, rabbits, dogs, pigs or primates. The animalmodel also can be used to determine the appropriate concentration rangeand route of administration. Such information then can be used todetermine useful doses and routes for administration in the animal to betreated, including humans.

Examples of other active agents, which can be included in thepharmaceutical composition or administered simultaneously orsequentially, in either order, with the pharmaceutical composition,include, but are not limited to, an anti-hypertensive agent (e.g.,adenosine, nifedipine, minoxidil, and magnesium sulfate), an angiogenicagent (e.g., VEGF, fibroblast growth factor (FGF), Sonic hedgehog (anindirect angiogenic agent), an SDF-1 mimetic (see, e.g., U.S. Pat. No.7,368,425), an IGD peptide (see, e.g., U.S. Pat. No. 7,232,802) ahydrazide compound (see, e.g., U.S. Pat. App. Pub. No. 2008/0274158), apro-angiogenesis, cytokine-stimulating peptide (see, e.g., U.S. Pat.App. Pub. No. 2008/0233081), an angiopoietin, such as angiopoietin-1,and the like. If the other active agent is administered simultaneouslyor sequentially, in either order, with the pharmaceutical composition,such as part of a separate pharmaceutical composition, desirably theother active agent is administered at such a time relative to theadministration of the pharmaceutical composition comprising ananti-sFlt-1 antibody to realize at least an additive, preferablysynergistic, effect.

The pharmaceutical composition comprising an anti-sFlt-1 antibody can beprovided as a therapeutic kit or pack. Individual components of the kitcan be packaged in separate containers, associated with which, whenapplicable, can be a notice in the form prescribed by a governmentalagency regulating the manufacture, use or sale of pharmaceuticals orbiological products, which notice reflects approval by the agency ofmanufacture, use or sale for human or animal administration. The kit canoptionally further contain one or more other active agents for use incombination with the pharmaceutical composition comprising ananti-sFlt-1 antibody. The kit can optionally contain instructions ordirections outlining the method of use or dosing regimen for thepharmaceutical composition comprising an anti-sFlt-1 antibody and/oradditional active agents or adjuvants.

When one or more components of the kit are provided as solutions, forexample an aqueous solution, or a sterile aqueous solution, thecontainer means can itself be an inhalant, syringe, pipette, eyedropper, or other such like apparatus, from which the solution can beadministered to a subject or applied to and mixed with the othercomponents of the kit.

The components of the kit also can be provided in dried or lyophilizedform, and the kit can additionally contain a suitable solvent forreconstitution of the lyophilized components. Irrespective of the numberor types of containers, the kit also can comprise an instrument forassisting with the administration of the composition to a patient. Suchan instrument can be an inhalant, a syringe, a pipette, a forceps, ameasuring spoon, an eye dropper, or a similar, medically approved,delivery vehicle. Accordingly, the pharmaceutical composition optionallycan be part of a kit comprising one or more containers in which theanti-sFlt-1 antibody, another active agent and/or the adjuvant can bepresent in the same or different containers.

Method of Prophylactic or Therapeutic Treatment

A method of treating a patient in therapeutic or prophylactic need of anantagonist of sFlt-1 is also provided. The method comprisesadministering to the patient a pharmaceutical composition comprising atherapeutically or prophylactically effective amount of an isolatedantibody, which specifically binds to sFlt-1 or a fragment thereof, andwhich has (i′) a variable heavy domain region comprising the amino acidsequence of SEQ ID NO: 2, (ii′) a variable light domain regioncomprising the amino acid sequence of SEQ ID NO: 4, or (iii′) a variableheavy domain region comprising the amino acid sequence of SEQ ID NO: 2and a variable light domain region comprising the amino acid sequence ofSEQ ID NO: 4. The composition further comprises a pharmaceuticallyacceptable carrier, diluent, and/or excipient. Optionally, thecomposition further comprises another active agent and/or an adjuvant.The method can prove useful in the treatment of preeclampsia,cardiovascular disease, and any disease, disorder or condition in whichthe inhibition of angiogenesis by sFlt-1 is undesirable, among others.

EXAMPLES

The following examples serve to illustrate the present disclosure. Theexamples are not intended to limit the scope of the claimed invention inany way.

Example 1

This example describes immunization of mice with human sFlt-1.

Female CAF1/J and RBF/DnJ mice (The Jackson Laboratory, Bar Harbor, Me.)were immunized six times with the sFlt-1 immunogen (sFlt-1 fused toC-terminal 6× histidine-tagged Fc region of human IgG and expressed inSf21 cells using a baculovirus expression system, R&D Systems,Minneapolis, Minn.). The adjuvant alternated between the Freund's(Difco, Detroit, Mich.) and MPL+TDM (Corixa, Hamilton, Mont.) AdjuvantSystems, beginning with Freund's Complete Adjuvant, which was used forthe primary inoculation. The inoculum was prepared by diluting theimmunogen in 0.9% sodium chloride and emulsifying with one of the twoadjuvants. At weeks 0, 3, 6, 8, 10 and 20 a 5 μg boost of the sFlt-1immunogen was administered to the mice. Three days prior to the fusion,the mice were administered a final boost of 20 μg sFlt-1 domains 1-3(Cell Sciences, Canton, Mass.). The immunogen was diluted in saline, andthe inoculum was injected directly into the spleen and the body cavitysurrounding the spleen for this final immunization.

Example 2

This example describes the screening of mice sera.

Sera samples, which were taken from the immunized mice 7-10 daysfollowing the final boost with immunogen, were tested in a 96-wellmicrotiter chemiluminescence immunoassay (CIA) for reactivity to sFlt-1.Assay plates (NUNC Corporation, Rochester, N.Y.) were coated with 100μL/well of rabbit anti-mouse IgG Fc antibody (Jackson Immuno Research,West Grove, Pa.) diluted to 5 μg/mL in phosphate-buffered saline (PBS).Plates were incubated overnight, and then the capture antibody wasremoved, followed by the addition of 200 μL/well fish gelatin diluted inPBS (block solution) for blocking. The plates were incubated and thenwashed with distilled water (dH₂O). Next, serial dilutions (in blocksolution) of the mouse sera or a positive control were added to theassay plates (100 μL/well), incubated and washed with dH₂O, Next,blocking solution (100 μL/well) or sFlt-1 (R&D Systems; diluted to 500ng/mL in block solution) was added to the assay plates, after which theassay plates were incubated and subsequently washed. The tracer,P1GF-ACR (placental growth factor linked to acridinium and diluted to 10ng/mL in block solution), was then added to the assay plates (100μL/well), after which the assay plates were incubated and subsequentlywashed. Finally, using a Microbeta Jet instrument (PerkinElmer, Waltham,Mass.), pre-trigger and trigger reagents (both Abbott Laboratories,Abbott Park, Ill.) were added, and the chemiluminescent signal was read.Samples were ranked based upon strength of signal.

Example 3

This example describes the production of hybridomas.

Mice were euthanized, and their spleens were harvested and placed intoHybridoma Serum-Free Medium (HSFM) (Invitrogen) supplemented only withL-glutamine (Invitrogen). A cell fusion was performed as described byKohler and Milstein (Nature 256: 495-497 (1975)). Each mouse spleen wasplaced into a separate petri dish containing HSFM with L-glutamine. Thesplenocytes were perfused out of each spleen using a syringe containingHSFM with L-glutamine and a cell scraper, and then counted using ahemocytometer. Splenocytes (6E6) from each mouse were pooled and mixedwith an equal number (1.8E7) of SP 2/0 myeloma cells and centrifugedinto a pellet. The fusion was accomplished by exposing the splenocytesand SP 2/0 cells to 50% polyethylene glycol (PEG) (molecular weight1300-1600, ATCC, Manassas, Va.) in HSFM. One mL of the PEG solution wasadded to the cell pellet over 30 seconds, followed by an additionalone-minute incubation. The PEG and cell pellet were diluted by slowlyadding 30 mL of HSFM with L-glutamine over 30 seconds. The fused cellswere then removed from suspension by centrifuging and subsequentlydecanting the supernatant. The cell pellet was re-suspended into a 50/50volume/volume mixture of HSFM with L-glutamine and spent media (i.e.,the media in which the myelomas were cultured in and which containscell-secreted growth factors). This media mixture was supplemented with15% fetal bovine serum (FBS; Hyclone Laboratories, Logan, Utah), HAT(Hypoxanthine, Aminopterin, Thymidine) (Sigma Laboratories, St. Louis,Mo.), HT supplement (Invitrogen), Hybridoma Cloning Factor (BioverisCorporation, Gaithersburg, Md.), and additional L-glutamine in order toselect for hybridomas. The cells were plated at 0.2 mL per well into96-well cell culture plates. At days 5 and 7 about one half of themedium in each well was removed by aspiration and replaced with HSFMsupplemented with 15% FBS, HT supplement, and L-glutamine. Hybridomaswere allowed to grow for 10 or 13 days, from the day of fusion, prior tosupernatant screening for antibody production.

Example 4

This example describes the screening and selection of hybridomas.

Samples of supernatants from hybridomas were analyzed for anti-sFlt-1antibodies using a CIA. Assay plates (NUNC) were coated with 100 μL/wellof rabbit anti-mouse IgG Fc antibody diluted to 5 μg/mL in PBS. Plateswere incubated overnight, the capture antibody was removed, and fishgelatin (diluted in PBS (block solution)) was added (200 μL/well) forblocking. The plates were incubated and then washed with dH₂O. Cellsupernatants (100 μL/well) were added to the blocked plates and allowedto incubate at room temperature for at least one hour. Next, sFlt-1antigen (250 ng/mL in blocking solution, 100 μL/well) was added to theassay plates, and the plates were incubated and subsequently washed withdH₂O. Next, P1GF-ACR (10 ng/mL in block solution, 100 μL/well) was addedto the assay plates, and the plates were incubated and subsequentlywashed. Finally, using a Microbeta Jet instrument, pre-trigger andtrigger reagents were added, and the chemiluminescent signal was read.Positives were selected based upon strength of signal. Positive hybridswere expanded to 24-well plates in HSFM supplemented with 1%L-glutamine, 10% FBS, and HT supplement. Following 3-7 days growth, the24-well cultures were again evaluated by CIA as described in thisexample, except multiple dilutions of antibody were tested forreactivity to both sFlt-1 and blocking solution (blocking solution usedas a control). Hybrids that demonstrated a relatively high reactivity tosFlt-1 in this assay were expanded to culture flasks. Hybrid 1-833 wascloned using a FACS (fluorescence-activated cell sorter) Aria instrument(Becton Dickinson, Franklin Lakes, N.J.). Parameters on the FACS wereset so that the instrument identified single cells and sorted them into96-well plates at 1 cell/well. The cells were cultured for 7-10 days inHSFM containing L-glutamine, FBS and HT, as previously described. Cellsupernatant was again tested by CIA for reactivity to sFlt-1 using themethod previously described. Clone 1-833-217 was identified assFlt-1-reactive, and cells were transferred to a 24-well cell cultureplate. Cells were then cultured in HSFM containing L-glutamine, FBS andHT for 3-5 days, after which the supernatant was tested (as described)and found to contain antibodies specifically reactive to sFlt-1. The1-833-217 cell line was weaned to growth in serum-free media (HSFMsupplemented only with L-glutamine) after which it was subcloned at 1cell/well via the FACS Aria (as described) into wells containing HSFMsupplemented with L-glutamine. Screening at the 96-well stage (asdescribed) identified subclone 1-833-527 as sFlt-1-reactive. Secondaryscreening at the 24-well stage (as described) confirmed that this clonewas specifically reactive to sFlt-1. A cell bank for 1-833-527 clone isstored in liquid nitrogen.

Example 5

This example describes competitive inhibition testing.

Testing was done to determine whether or not the 1-833-217 antibodybinds to the same epitope on sFlt-1 as previously existing, commerciallyavailable antibodies. Assay plates (NUNC) were coated with 100 μL/wellof P1GF (R&D Systems) diluted to 500 ng/mL in PBS and incubatedovernight. The P1GF was removed, and plates were blocked with 200μL/well blocking solution (bovine serum albumin (BSA) diluted in PBS).The plates were then incubated for about 30 minutes and washed withdH₂O, Next, sFlt-1 (diluted to 500 ng/mL in PBS) was added to each assaywell (100 μL/well), and the plates were incubated for 2-3 hours and thensubsequently washed with dH₂O, Next, unlabeled antibodies (diluted to 10μg/mL in PBS) were added to the appropriate assay wells (100 μL/well)and incubated for 2-3 hours. Next, 50 μL of biotin-labeled 1-833-217antibody, diluted to 250 ng/mL in blocking solution, were added to eachassay well (note that the unlabeled antibody was not removed prior toadding the biotin-labeled antibody). The plates were incubated for 10minutes, the reagents were removed, and the plates were washed withdH₂O. Next, 100 μL of peroxidase-conjugated streptavidin (JacksonImmunoResearch), diluted to 200 ng/mL in blocking solution, were addedto each assay well. The plates were incubated for about 30 minutes andthen washed with dH₂O. O-phenylenediamine substrate (OPD) was used asthe chromagen to generate signal. The reaction was quenched using 1 Nsulfuric acid. Signal was read at a wavelength of 492 nm. Results fromthe assay indicated that the 1-833-217 clone binds to a differentepitope on sFlt-1 vs. either of the two commercially availableantibodies, i.e., clone 43 (R&D Systems) and clone 321 (catalog no.MAB321, hereafter “monoclonal antibody 321,” R&D Systems).

Example 6

This example describes the characterization of the affinities/kineticsof anti-sFlt-1 monoclonal antibodies for sFlt-1 antigen (domains 1-3).The affinities/kinetics of anti-human sFlt-1 monoclonal antibodies1-654-302, 1-833-217, and 1-833-527 for sFlt-1 antigen (domains 1-3; R&DSystems) were determined using a BIAcore 2000 instrument (BIAcore, GEHealthcare, Piscataway, N.J.). First, a ˜5,000 RU rabbit anti-mouse IgGCapture Biosensor was created by amine-coupling rabbit anti-mouse IgGantibody (BIAcore, GE Healthcare) to a CM4 biosensor chip (BIAcore, GEHealthcare) via EDC/NHS/ethanolamine chemistry provided in an AmineCoupling Kit (BIAcore, GE Healthcare). sFlt-1 antibody and sFlt-1antigen (domains 1-3) were diluted into a running buffer (hereinafter“running buffer”) composed of HBS-EP buffer (BIAcore, GE Healthcare)spiked with 1% BSA, 1% CM-Dextran, and 0.1% Tween 20. Each sFlt-1antibody was diluted to 1 μg/mL, and sFlt-1 antigen (domains 1 through3) was diluted to concentrations ranging from 2.56 to 250 nM using a2.5-fold dilution series. After equilibrating the rabbit anti-mouse IgGCapture Biosensor for 5 minutes at 10 μL/minute with running buffer,sFlt-1 antibody (9-14 μL) was injected over individual flow cells withone flow cell being left blank as a reference flow cell. The flow cellswere washed for 5 minutes at 60 μL/minute with running buffer. Then 150μL of sFlt-1 antigen at a random concentration were injected across thebiosensor. The biosensor was subsequently washed for 6 minutes at 60μl/minute with running buffer. The biosensor was regenerated with one 30μL injection of 10 mM glycine, pH 1.7 (BIAcore, GE Healthcare), at aflow rate of 10 μL/minute. All concentrations of sFlt-1 antigen weretested in duplicate. The binding kinetics (association and dissociation)were monitored via sensorgrams. The sensorgrams were double-referencedand fit to a 1:1 binding model using Scrubber 2.0 software (BioLogicSoftware Pty Ltd., Australia) to determine association and dissociationrates, as well as overall K_(D). The results are shown in Table 1.

TABLE 1 sFlt-1 monoclonal antibody k_(on) (M⁻¹s⁻¹) k_(off) (s⁻¹) K_(D)(M) 1-654-302 1.712(4) × 10⁶ 4.9(1) × 10⁻⁴ 2.87(6) × 10⁻¹⁰ 1-833-2171.936(5) × 10⁶ 4.1(1) × 10⁻⁴ 2.14(7) × 10⁻¹⁰ 1-833-527 1.895(6) × 10⁶4.0(2) × 10⁻⁴ 2.13(9) × 10⁻¹⁰

Standard errors of determined values are reported in parentheses withrespect to the smallest number place value.

Example 7

This example describes the screening of various anti-sFlt-1 monoclonalantibodies in a double monoclonal antibody sandwich assay.

Nineteen monoclonal antibodies obtained in accordance with the methodsset forth herein and 4 monoclonal antibodies obtained from R&D Systemswere screened using a chemiluminescent microparticle immunoassayemploying the ARCHITECT® automated analyzer (Abbott Laboratories; see,also, U.S. Pat. Nos. 5,795,784 and 5,856,194, both of which areincorporated herein by reference in their entireties). An sFlt-1 antigen(sFlt-1 domains 1-3 from mouse myeloma NSO, sFlt-1 domains 1-3:Fcchimera from mouse myeloma NSO, or sFlt-1 domains 1-6:Fc chimera fromSf21 cells using baculovirus) was contacted with a microparticlereagent, which contained paramagnetic, streptavidin-coatedmicroparticles coated (300 ng/mL) with biotin-labeled anti-sFlt-1monoclonal antibody or paramagnetic microparticles coated withanti-sFlt-1 monoclonal antibody chemically attached to themicroparticles with EDAC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide,hydrochloride), and allowed to react for 18 minutes at 37° C. on theARCHITECT® analyzer. After the 18-minute reaction, the microparticleswere washed to eliminate any unbound sFlt-1 antigen, CPSP-conjugated orSPSP-conjugated anti-sFlt-1 monoclonal antibody (300 ng/mL) was added,and the sample was incubated for four minutes on the ARCHITECT®analyzer. The CPSP/SPSP-conjugated anti-sFlt-1 monoclonal antibody boundto the sFlt-1 antigen already bound to the anti-sFlt-1 monoclonalantibody coated on the microparticles. After four minutes, the unboundconjugate was eliminated by washing, and pre-trigger and triggersolutions were added to cause a chemiluminescent reaction, which wasmeasured in relative light units (RLUs). A sandwich was considered to bepositive if the RLU for the calibrator at 1,000 pg/mL was greater than400,000.

The results are set forth in FIG. 3. Anti-sFlt-1 monoclonal antibody1-780-103 and 2-106-105 coated on microparticles formed sandwiches witha number of other antibodies. Anti-sFlt-1 monoclonal antibody 1-780-103and R&D Systems' monoclonal antibody 321, 49508 and 49566 may have fast“on” rates and can form sandwiches with multiple antibodies. Anti-sFlt-1monoclonal antibody 1-330-202 and 1-333-205 and R&D Systems' 49508 reactwith sFlt-1 domains 1-6:Fc chimera only and may bind to a region outsideof domains 1-3. Anti-sFlt-1 monoclonal antibody 2-106-105 may have aslow “on” rate and cannot be used as the conjugate. Anti-sFlt-1monoclonal antibodies, which cannot form sandwiches with P1GF-acridiniummay be used to detect free sFlt-1 (like 49566). Anti-sFlt-1 monoclonalantibody 1-330-202, 1-333-205, and 2-154-307 and R&D Systems' 49511 haveno or limited reactivity.

Example 8

This example describes the use of various combinations of anti-sFlt-1monoclonal antibodies in the detection of total sFlt-1 and free sFlt-1.

A composition comprising a fixed amount of sFlt-1 was prepared. Varyingamounts of P1GF (0-100 molar excess), which complexes with sFlt-1, wereadded to separate aliquots of the sFlt-1 composition. Variouscombinations of monoclonal antibodies were assayed for their ability todetect free sFlt-1 (i.e., sFlt-1 that is not complexed with P1GF) andtotal sFlt-1 (i.e., sFlt-1 that is complexed with P1GF and free sFlt-1).A chemiluminescent microparticle immunoassay employing the ARCHITECT®automated analyzer as described in Example 7 was used. In one format,the microparticles (0.10% solid) were coated with monoclonal antibody1-833-527, and the R&D Systems monoclonal antibody 321 was used as theconjugate (the 527/321 format). In another format, the microparticles(0.05% solid) were coated with the R&D Systems monoclonal antibody49566, and the R&D Systems monoclonal antibody 321 was used as theconjugate (the 566/321 format). In yet another format, themicroparticles (0.10% solid) were coated with the R&D Systems monoclonalantibody 321, and P1GF was used as the conjugate (the 321/P1GF format).The results, expressed in RLUs and relative RLUs, are set forth in Table2.

TABLE 2 Format [PIGF] RLUs Relative RLUs nM 527/321 566/321 321/PIGF527/321 566/321 321/PIGF 0.0 1104966 745206 1126081 1.00 1.00 1.00 0.21093205 742865 1048390 0.99 1.00 0.93 0.4 1090443 743200 976763 0.991.00 0.87 0.8 1045042 718955 838054 0.95 0.96 0.74 1.6 1036661 646194621663 0.94 0.87 0.55 3.2 1012088 506651 382994 0.92 0.68 0.34 6.41032870 420755 274102 0.93 0.56 0.24 12.9 1014395 359070 226242 0.920.48 0.20 25.8 1030129 302275 191487 0.93 0.41 0.17 51.5 1032753 248866172991 0.93 0.33 0.15 103.0 1028819 205840 166618 0.93 0.28 0.15 206.01029538 183671 157486 0.93 0.25 0.14Thus, these findings confirm that the 527/321 format enabled an assayfor total sFlt-1.

Example 9

This example describes the sequencing of the anti-sFlt-1 monoclonalantibody 1-833-527.

mRNA was extracted from hybridoma cell cultures using commerciallyavailable reagents (Oligotex direct mRNA kit, Qiagen, Inc., Valencia,Calif.) following the manufacturer's recommendations. IgG heavy chaincDNA and kappa light chain cDNA were generated from the extracted mRNAusing commercially available murine Ig primers, MuIgGVH3′-2 andMuIgkVL3′-1, respectively (Mouse Ig-Primer set, Novagen, EMD Chemicals,Inc., Gibbstown, N.J.), following standard protocols. The variable heavy(VH) and variable light (VL) genes were then amplified from theirrespective cDNA using polymerase chain reaction (PCR) and pools of IgG-and IgK-specific primers from the same commercially available murine Igprimer kits referenced above using standard protocols. Amplified VH andVL PCR products were cloned into a commercially available vector(pCR2.1-TOPO cloning kit, Qiagen, Inc.) per the manufacturer'sdirections and transformed into E. coli. Sequence analysis was performed(BigDye Terminator v3.1 cycle sequencing kit, Applied Biosystems, FosterCity, Calif.) on plasmids isolated from multiple transformed E. colicolonies to identify the VH and VL gene sequences. The sequences are setforth in FIGS. 1-2. FIG. 1 shows the nucleotide (SEQ ID NO: 1) and aminoacid (SEQ ID NO: 2) sequences of the variable heavy chain (VH) regionsof the anti-sFlt-1 monoclonal antibody 1-833-527. FIG. 2 sets forth thenucleotide (SEQ ID NO: 3) and amino acid (SEQ ID NO: 4) sequences of thevariable light chain (VL) regions of the anti-sFlt-1 monoclonal antibody1-833-527.

Furthermore, sequencing data confirmed that the anti-sFlt-1 monoclonalantibody 1-833-527 had the identical nucleotide sequence as theanti-sFlt-1 monoclonal antibody 1-833-217 described in Example 4.

Example 10

This example describes the reference range of sFlt-1 in an apparentlyhealthy population.

An immunoassay employing the anti-sFlt-1 monoclonal antibody 1-833-527immobilized on paramagnetic microparticles as the capture reagent andacridinium labeled R&D Systems anti-sFlt-1 monoclonal antibody 321 asthe detection reagent was used to test specimens on the ARCHITECT®analyzer as described in Example 7. R&D Systems sFlt-1 antigen domains1-3:Fc chimera was used as calibrator material. A calibrator setconsisting of concentrations of 0, 125, 400, 2,500, 12,000 and 50,000pg/mL was used to calibrate the assay. The functional sensitivity of theassay in this configuration provided a 10% CV at a concentration lessthan 90 pg/mL and a 20% CV at a concentration less than 20 pg/mL. (The %Coefficient of Variation is (CV) is defined as the standard deviation ofa measurement (SD) divided by the mean analyte concentration andmultiplied by 100.)

Following institutional review board approval and informed consent fromthe subjects, serum and EDTA plasma was obtained from 305 apparentlyhealthy volunteers between ages 18 and 81 years with no history ofcardiac disease, diabetes, or hypertension. The plasma specimens wereplaced on ice after collection and processed within 4 to 6 hours. Theserum specimens were allowed to clot at ambient temperature for 30minutes, then placed on ice and processed within 4 to 6 hours. Afterprocessing, the serum and plasma were aliquoted and frozen at less than−60 degrees C. or colder. Prior to testing on the ARCHITECT® analyzer,the specimens were thawed and centrifuged at greater than 2,000×g for atleast 10 minutes.

Descriptive statistics for sFlt-1 (pg/mL) concentrations in thispopulation are described in Tables 3 and 4 below. The medianconcentrations in the two specimen types are equivalent across genderand age groups. The median of the serum specimens is approximately 50pg/mL or 20% higher than the median observed in EDTA plasma.

TABLE 3 EDTA Plasma All Male Female Age ≧40 Age <40 Number (N) 303 126177 169 134 Mean 252.9 256.6 250.3 258.3 246.1 Standard Deviation 38.234.3 40.6 40.2 34.3 Minimum 160.6 179.4 160.6 160.6 172.0 Maximum 412.6351.4 412.6 412.6 344.9 Quantiles  5% 195.1 202.8 187.3 199.5 189.5 10%205.7 215.1 199.3 208.4 199.3 25% 227.4 234.4 225.6 235.0 224.2 50%(Median) 248.8 251.4 246.0 250.8 244.6 75% 275.8 280.2 274.0 281.3 269.390% 304.2 307.2 298.4 317.9 290.2 95% 322.9 322.1 325.8 332.9 306.3

TABLE 4 Serum All Male Female Age ≧40 Age <40 Number (N) 305 127 178 168137 Mean 304.0 305.9 302.7 308.7 298.3 Standard Deviation 42.3 38.2 45.043.9 39.5 Minimum 212.3 231.4 212.3 212.3 225.6 Maximum 444.9 444.9439.6 444.9 412.3 Quantiles  5% 239.7 247.8 238.8 245.5 236.7 10% 252.5257.1 250.3 257.6 249.0 25% 272.9 276.9 269.2 278.6 268.3 50% (Median)299.9 303.3 299.3 301.9 298.1 75% 331.3 331.1 331.6 331.0 331.5 90%358.2 354.8 360.2 364.0 352.4 95% 380.2 367.1 391.3 400.1 360.7

Example 11

This example describes the use of an exemplary sFlt-1 assay as describedherein in a patient population at risk for cardiovascular events.

Specimens from a prospective, blinded, multi-center cohort clinicalstudy were evaluated using the ARCHITECT® sFlt-1 assay described inExample 10. Patients in the study were recruited at presentation to theemergency department with chest pain and signs/symptoms of ischemiasuggestive of acute coronary syndrome (ACS).

Institutional review boards at each site approved the study protocol,and informed consent was obtained from all subjects. Data regardingclinical characteristics, cardiac procedures and cardiac events duringhospitalization were collected. Telephone follow up occurred at 30 daysand approximately one year after enrollment to assess for major adversecardiac events (MACE) which consisted of death, myocardial infarction,or revascularization.

Patient blood samples were obtained in lithium heparin plasma, EDTAplasma, and serum collection tubes at enrollment (0 hours), 4 to 8 hourslater, and if still hospitalized, 12 to 16 hours after enrollment. Bloodwas collected and processed according to local site procedures. Sampleswere stored centrally at less than −60 degrees C. prior to analysis.Prior to testing on the ARCHITECT® analyzer, the specimens were thawedand centrifuged at greater than 2,000×g for at least 10 minutes.

Serum specimens from a total of 497 patients were available forevaluation in the sFlt-1 assay. Lithium heparin plasma specimens wereused for evaluation in the commercially available ARCHITECT® Troponin Iassay. There were 80 patients (16.1%) in this group that experiencedMACE between presentation to the emergency department and the one yearfollow up. The descriptive characteristics of sFlt-1 are shown below inTable 5. A significant increase in sFlt-1 was observed in the patientsthat experienced MACE compared to those that did not have a cardiacevent. The Wilcoxon test was used to determine statistical significance(p-value <0.0001) between the median concentrations of the patients withand without MACE.

TABLE 5 Descriptive characteristics of sFlt-1 (pg/mL) in the clinicalstudy. Total Patients without Patients with Patients MACE MACE (N = 497)(N = 417) (N = 80) Mean 936.3 770.9 1798.4 Standard Deviation 3050.53011.8 3124.6 Minimum 149.8 149.8 215.5 Maximum 40646.6 40646.6 17863.5Quantiles 25% 267.9 264.1 317.5 50% (median) 310.9 301.9 418.1 75% 392.6359.9 1391.8

Further analysis of the clinical data was made by stratifying patientsusing the upper limit of the sFlt-1 reference interval in serumspecimens (95th percentile, 380 pg/mL) and the ARCHITECT® Troponin I atthe 99th percentile (0.028 ng/mL) as stated in the package insert. Thereare 424 patients with Troponin I values less than the 99th percentileand 38 (9.0%) of these patients experienced MACE. Of the 424 Troponin Inegative patients; there are 87 patients with sFlt-1 concentrationgreater than the 95th percentile with a 19.5% incidence of MACE comparedto a 6.2% incidence of MACE in the 337 patients that are sFlt-1 negative(see FIG. 4). The difference in event rates observed when the Troponin Inegative group is stratified by sFlt-1 is statistically significant(p-value equal to 0.0003) using the Fisher's Exact test.

Example 12

This example describes the use of an exemplary sFlt-1 assay as describedherein to assess sFlt-1 in a population of pregnant women.

Specimens from a prospective, blinded, multi-center cohort clinicalstudy were evaluated using the ARCHITECT® sFlt-1 assay described inExample 10. Institutional review boards at each site approved the studyprotocol and informed consent was obtained from all subjects. Subjectswere enrolled in the study at estimated gestational age less than orequal to 15 weeks. Serum and EDTA plasma were collected at enrollment,additional specimens were collected at approximately 16-20 weeks, 24-28weeks, and 34-38 weeks gestational age if still pregnant. At each visitblood pressure, urinary protein, and weight was recorded as part ofstandard clinical care. For hypertensive patients, the specifichypertensive condition suggested by the patient's medical conditions wascategorized based on the American College of Obstetricians andGynecologists recommendations pertaining to preeclampsia. An additionalspecimen was collected as an option in subjects diagnosed withpreeclampsia between the time of diagnosis and delivery. Blood wascollected and processed according to local site procedures. Samples werestored centrally at less than −60 degrees C. prior to analysis. Prior totesting on the ARCHITECT® analyzer, the specimens were thawed andcentrifuged at greater than 2,000×g for at least 10 minutes.

To measure pregnancy samples, a calibrator providing as assay range from0 to 150 ng/mL was employed consisting of concentrations of 0, 2.5, 10,35, 75 and 150 ng/mL to calibrate the assay. EDTA plasma specimens(n=2,458) from a total of 653 patients were available for evaluation inthe ARCHITECT® sFlt-1 assay. The sFlt-1 values were plotted against thegestational age for each specimen as shown in FIG. 5, a rise in sFlt-1concentration of approximately 20% is observed with increasinggestational age.

The specimens (n=2,453) were also evaluated in the commerciallyavailable R&D Systems Human sVEGF R1 (sFlt-1) ELISA assay (Catalog#DVR100). The correlation is shown in FIG. 6, linear regression resultsin the fit: R&D Systems sFlt-1=0.223*(ARCHITECT sFlt-1)+0.571 with aSpearman correlation coefficient, r=0.897. The sample concentrationsranged from 0.1 to 91.0 ng/mL on the ARCHITECT sFlt-1 assay.

All of the data points in FIGS. 5 and 6 are represented as open circles;regions that appear to be solid are due to overlap in data points in thelarge sample sets.

All patents, patent application publications, journal articles,textbooks, and other publications mentioned in the specification areindicative of the level of skill of those in the art to which thedisclosure pertains. All such publications are incorporated herein byreference to the same extent as if each individual publication werespecifically and individually indicated to be incorporated by reference.

The invention illustratively described herein may be suitably practicedin the absence of any element(s) or limitation(s), which is/are notspecifically disclosed herein. Thus, for example, each instance hereinof any of the terms “comprising,” “consisting essentially of,” and“consisting of” may be replaced with either of the other two terms.Likewise, the singular forms “a,” “an,” and “the” include pluralreferences unless the context clearly dictates otherwise. Thus, forexample, references to “the method” includes one or more methods and/orsteps of the type, which are described herein and/or which will becomeapparent to those ordinarily skilled in the art upon reading thedisclosure.

The terms and expressions, which have been employed, are used as termsof description and not of limitation. In this regard, where certainterms are defined under “Definitions” and are otherwise defined,described, or discussed elsewhere in the “Detailed Description,” allsuch definitions, descriptions, and discussions are intended to beattributed to such terms. There also is no intention in the use of suchterms and expressions of excluding any equivalents of the features shownand described or portions thereof. Furthermore, while subheadings, e.g.,“Definitions,” are used in the “Detailed Description,” such use issolely for ease of reference and is not intended to limit any disclosuremade in one section to that section only; rather, any disclosure madeunder one subheading is intended to constitute a disclosure under eachand every other subheading.

It is recognized that various modifications are possible within thescope of the claimed invention. Thus, it should be understood that,although the present invention has been specifically disclosed in thecontext of preferred embodiments and optional features, those skilled inthe art may resort to modifications and variations of the conceptsdisclosed herein. Such modifications and variations are considered to bewithin the scope of the invention as defined by the appended claims.

1.-2. (canceled)
 3. A method of determining the presence, amount orconcentration of sFlt-1 or a fragment thereof in a test sample, whichmethod comprises assaying the test sample for sFlt-1 (or a fragmentthereof) by an immunoassay employing at least one antibody and at leastone detectable label and comprising comparing a signal generated by thedetectable label as a direct or indirect indication of the presence,amount or concentration of sFlt-1 (or a fragment thereof) in the testsample to a signal generated as a direct or indirect indication of thepresence, amount or concentration of sFlt-1 (or a fragment thereof) in acontrol or a calibrator, which is optionally part of a series ofcalibrators in which each of the calibrators differs from the othercalibrators in the series by the concentration of sFlt-1 (or a fragmentthereof), wherein one of the at least one antibody is an isolatedantibody, which specifically binds to sFlt-1 (or a fragment thereof) andwhich has (i) a variable heavy domain region comprising the amino acidsequence of SEQ ID NO: 2, (ii) a variable light domain region comprisingthe amino acid sequence of SEQ ID NO: 4, or (iii) a variable heavydomain region comprising the amino acid sequence of SEQ ID NO: 2 and avariable light domain region comprising the amino acid sequence of SEQID NO: 4, whereupon the presence, amount or concentration of sFlt-1 (ora fragment thereof) in the test sample is determined.
 4. The method ofclaim 3, wherein the method comprises the following steps: (i)contacting the test sample with at least one capture antibody, whichbinds to an epitope on sFlt-1 (or a fragment thereof) so as to form acapture antibody/sFlt-1 (or a fragment thereof) complex, (ii) contactingthe capture antibody/sFlt-1 (or a fragment thereof) complex with atleast one detection antibody, which comprises a detectable label andbinds to an epitope on sFlt-1 (or a fragment thereof) that is not boundby the capture antibody, to form a capture antibody/sFlt-1 (or afragment thereof)/detection antibody complex, and (iii) determining thepresence, amount or concentration of sFlt-1 (or a fragment thereof) inthe test sample based on the signal generated by the detectable label inthe capture antibody/sFlt-1 (or a fragment thereof)/detection antibodycomplex formed in (ii), whereupon the presence, amount or concentrationof sFlt-1 (or a fragment thereof) in the test sample is determined. 5.The method of claim 3, wherein the method comprises the following steps:(i) contacting the test sample with at least one capture antibody, whichbinds to an epitope on sFlt-1 (or a fragment thereof) so as to form acapture antibody/sFlt-1 (or a fragment thereof) complex, andsimultaneously or sequentially, in either order, contacting the testsample with detectably labeled sFlt-1 (or a fragment thereof), which cancompete with any sFlt-1 (or a fragment thereof) in the test sample forbinding to the at least one capture antibody, wherein any sFlt-1 (or afragment thereof) present in the test sample and the detectably labeledsFlt-1 compete with each other to form a capture antibody/sFlt-1 (or afragment thereof) complex and a capture antibody/detectably labeledsFlt-1 (or a fragment thereof) complex, respectively, and (ii)determining the presence, amount or concentration of sFlt-1 in the testsample based on the signal generated by the detectable label in thecapture antibody/detectably labeled sFlt-1 (or a fragment thereof)complex formed in (ii), wherein the signal generated by the detectablelabel in the capture antibody/detectably labeled sFlt-1 (or a fragmentthereof) complex is inversely proportional to the amount orconcentration of sFlt-1 in the test sample, whereupon the presence,amount or concentration of sFlt-1 in the test sample is determined. 6.The method of claim 3, which further comprises simultaneously orsequentially, in either order, determining the amount or concentrationof vascular endothelial growth factor (VEGF) (or a fragment thereof)and/or placental growth factor (P1GF) (or a fragment thereof) in thetest sample, which method comprises assaying the test sample for VEGF(or a fragment thereof) and/or P1GF (or a fragment thereof) by an assayemploying at least one specific binding partner for VEGF (or a fragmentthereof) and/or at least one specific binding partner for P1GF (or afragment thereof), respectively, and at least one detectable label andcomprising a signal generated by the detectable label as a direct orindirect indication of the amount or concentration of VEGF (or afragment thereof) and/or P1GF (or a fragment thereof) in the test sampleto a signal generated as a direct or indirect indication of the amountor concentration of VEGF (or a fragment thereof) and/or P1GF (or afragment thereof), respectively, in a control or calibrator, which isoptionally part of a series of calibrators in which each of thecalibrators differs from the other calibrators in the series by theconcentration of VEGF or P1GF, respectively, whereupon the amount orconcentration of VEGF (or a fragment thereof) and/or P1GF (or a fragmentthereof) in the test sample is determined.
 7. The method of claim 4,which further comprises simultaneously or sequentially, in either order,determining the amount or concentration of VEGF (or a fragment thereof)and/or P1GF (or a fragment thereof) in the test sample, which methodcomprises assaying the test sample for VEGF (or a fragment thereof)and/or P1GF (or a fragment thereof) by an assay employing at least onespecific binding partner for VEGF (or a fragment thereof) and/or atleast one specific binding partner for P1GF (or a fragment thereof),respectively, and at least one detectable label and comprising comparinga signal generated by the detectable label as a direct or indirectindication of the amount or concentration of VEGF (or a fragmentthereof) and/or P1GF (or a fragment thereof) in the test sample to asignal generated as a direct or indirect indication of the amount orconcentration of VEGF and/or P1GF, respectively, in a control orcalibrator, which is optionally part of a series of calibrators in whicheach of the calibrators differs from the other calibrators in the seriesby the amount concentration of VEGF or P1GF, respectively, whereupon theamount or concentration of VEGF (or a fragment thereof) and/or P1GF (ora fragment thereof) in the test sample is determined.
 8. The method ofclaim 5, which further comprises simultaneously or sequentially, ineither order, determining the amount or concentration of VEGF (or afragment thereof) and/or P1GF (or a fragment thereof) in the testsample, which method comprises assaying the test sample for VEGF (or afragment thereof) and/or P1GF (or a fragment thereof) by an assayemploying at least one specific binding partner for VEGF (or a fragmentthereof) and/or at least one specific binding partner for P1GF (or afragment thereof), respectively, and at least one detectable label andcomprising comparing a signal generated by the detectable label as adirect or indirect indication of the amount or concentration of VEGF (ora fragment thereof) and/or P1GF (or a fragment thereof) in the testsample to a signal generated as a direct or indirect indication of theamount or concentration of VEGF and/or P1GF, respectively, in a controlor calibrator, which is optionally part of a series of calibrators inwhich each of the calibrators differs from the other calibrators in theseries by the concentration of VEGF or P1GF, respectively, whereupon theamount or concentration of VEGF (or a fragment thereof) and/or P1GF (ora fragment thereof) in the test sample is determined.
 9. The method ofclaim 3, wherein the test sample is from a patient and the methodfurther comprises diagnosing, prognosticating, or assessing the efficacyof therapeutic/prophylactic treatment of the patient, wherein, if themethod further comprises assessing the efficacy oftherapeutic/prophylactic treatment of the patient, the method optionallyfurther comprises modifying the therapeutic/prophylactic treatment ofthe patient as needed to improve efficacy.
 10. The method of claim 4,wherein the test sample is from a patient and the method furthercomprises diagnosing, prognosticating, or assessing the efficacy oftherapeutic/prophylactic treatment of the patient, wherein, if themethod further comprises assessing the efficacy oftherapeutic/prophylactic treatment of the patient, the method optionallyfurther comprises modifying the therapeutic/prophylactic treatment ofthe patient as needed to improve efficacy.
 11. The method of claim 5,wherein the test sample is from a patient and the method furthercomprises diagnosing, prognosticating, or assessing the efficacy oftherapeutic/prophylactic treatment of the patient, wherein, if themethod further comprises assessing the efficacy oftherapeutic/prophylactic treatment of the patient, the method optionallyfurther comprises modifying the therapeutic/prophylactic treatment ofthe patient as needed to improve efficacy.
 12. The method of claim 3,wherein the method is adapted for use in an automated system or asemi-automated system.
 13. The method of claim 4, wherein the method isadapted for use in an automated system or a semi-automated system. 14.The method of claim 5, wherein the method is adapted for use in anautomated system or a semi-automated system. 15.-21. (canceled)